WELCOME BACK TO DAY 2 OF THE
THIS FANTASTIC MEETING.
IT'S MY PLEASURE TO INTRODUCE
DR. SONG WHO IS OUR FIRST
SPEAKER TODAY FOR A SHORT TALK.
WELCOME.
>> THANK YOU.
THANK YOU NIKI, THANK YOU PAM
FOR DOING THESE FOR ALL THESE
YEARS.
DID I HEAR AN ECHO?
ALL RIGHT.
OKAY, RIGHT HERE.
ALL RIGHT, SO I'M GOING TO--IT'S
NOT--OKAY.
SO I'M GOING TO TALK ABOUT THE
GRANT WE WERE AWARDED AND HAVE
BYE-BYE WORKING ON FOR SEVERAL
YEARS, SO IT'S A FOSSIL CHEMICAL
METHOD TO AN ISOLATE GLYCAN TO
ADDRESS THE LACK OF GLYCANS IN A
DIFFERENT WAY, SO I DON'T HAVE
TO SHOW THESE FOR THE WHOLE
AUDIENCE BUT THERE ARE MANY,
MANY DIFFERENT KIND OF GLYCANS
BUT THEY OCCUR MOSTLY AS THE
GLYCAN CONJUGATES INSTEAD OF
FREE PRODUCING GLYCANS.
SO IF WE--OH, SORRY.
THIS 1.
SO WE ALL AGREE, AT LEAST IN
THIS COMMUNITY THAT 1 OF THE
MAJOR BARRIER IF IT'S NOT THE
ONLY 1, BUT THE 1 OF THE MAJOR
BARRIER IS THE LACK OF--OH.
READILY RELEVANT CARBOHYDRATES.
SO HOW DO WE GET THESE?
THE COMMUNITY HAS BEEN WORKING
ON THESE VERY DILIGENTLY, SO
THESE ARE RELEVANT DPLI
KAN--KANAS, IT'S BASICALLY 2
WAYS, EITHER THROUGH VETTING
POSSIBLE, OR THROUGH THE NATURAL
PRODUCTS ISOLATION, SEPARATION.
SO, FOR--I AM GOING TO USE THE
OTHER 1.
YEAH, SO CHEMICAL, HAVE YOU
HEARD YESTERDAY, A VERY
IMPRESSIVE TALKS FROM
DR. WILSON, AND DR. JAN LUOT
SYNTHESIS OF IMMUNO GLYCANS, BUT
YOU WILL HEAR A LOT MORE ON
THE--SYNTHESIS OF OTHER CLASS OF
GLYCANS, BUT WHAT WE'RE GOING TO
FOCUS ON IS THE WAY TO ISOLATE
AND SEPARATE THE NATURAL
GLYCANS.
SO AS WE--OKAY, SO CAN WE REALLY
PRODUCE LARGER AMOUNT OF GLYCAN
FROM MEASURABLE SOURCES, SO UP
UNTIL NOW, THERE'S PRODUCING
NATURAL GLYCANS FOR REASON HAS
BEEN IN THE GRIEK O BIOLOGY AREA
FOR AT LEAST AS LONG AS
SYNTHESIS, SO, IT'S--IT'S NOT A
PROBLEM TO GET A SMALL AMOUNT OF
A GLYCAN.
WE HAVE ENZYMES, WE HAVE SOME
CHEMICALS THAT CAN BE USED TO
RELEASE GLYCANS, BUT THERE ARE
SEVERAL PROBLEM OF THESE--EXCUSE
ME,--VERY SENSITIVE.
SO THE LOW AMOUNT OF NATURAL
MATERIALS, AND THERE'S NO
SCALABLE PROCESS TO RELEASE GLUE
MARIOUS ANS AND THE GLYCANS
WHATEVER YOU RELEASE IS A
HETEROGENIUS DPLI KAN--KANA MIX
TOUR THAT REQUIRES SEPARATION,
SO WHAT WE WANT TO ADDRESS IS
WEATHER WE CAN ACTUALLY LOOK AT
AND TO DEAL WITH A LARGER AMOUNT
OF NATURAL MATERIAL SO WE CAN
GET LARGER AMOUNT OF NATURAL GUY
KAN--KANAS SO A FEW YEARS AGO, I
NOTICED THIS PAPER IF YOU HAVE
YOU CANOXIDATE THIS TO A TRIAL.
SO WE'RE THINKING IF YOU HAVE A
GLYCO PROTEIN, WE KNOW PRONAIS
CAN TO GET THE LAST GLYCO IMMUNO
ASSAY, SO WHAT WE WILL GET IF WE
ORIGINAL REDUCING GLYCANS IF YOU
HEATED THE MIXTURE UP, BUT THIS
METHOD IS NOT IDEAL BECAUSE IT'S
STILL INVOLVED ENZYME IS IT'S
REALLY YOU CAN IMAGINE DOING
THIS WITH A HUNDRED GRAMS OF
MATERIAL MATERIAL BUT YOU CANNOT
WORK WITH KILOGRAMS OF MATERIAL
AND ALSO THIS THING IS STILL
CHEMICAL BY ITSELF DOESN'T
WELCOMER ON LIKE UNTREATED
MATERIAL AND ALSO IT DOESN'T
WORK ON O-GLUE MARIOUS ANS AND
THE LIPIDS SO WE WERE LOOKING
BUT WE WERE ENCOURAGED BY
THEOXIDATIVE DEVIATION PATHWAY,
SO WE WERE LOOKING FOR OTHEROX
DABTS, SO THE FIRST THING MEAN
WE TRY, ACTUALLY WORKED WHICH IS
SODIUM HYDROCHLORIDE, WHICH IS
PROBABLY THE SIMPLEST INOX DACE
IN EVERYONE'S HOME, IT'S THE
HOUSEHOLD BLEACH.
SO, YOU TREAT A GLYCO PROTEIN
WITH BLEACH, IS THEY ARE
RELEASED, THE PROTEINS DEGRADED
AND THE PROTEINS MOVE ON THE
PLATE AND THEY VIEW--IF YOU LOOK
AT THE MASS SPEC, CAN YOU SEE A
LOT OF REDUCING GLYCANS HERE AND
OF COURSE, THE SCHEME IS LIKE
THESE, FOR NGLYCAN PROTEINS THEN
WHAT YOU GET IS A FREE REDUCING
GLYCAN.
THIS DIRECTLY TREATS THE GLYCO
PRACTICES TEEN, WE DON'T NEED
ANY PROTEIN TREATED WITH ANY
ENZYME TO BREAK THIS APART.
AND THIS ALSO WORKS WITH
[INDISCERNIBLE] AS LONG AS YOU
HOMOGINIZE IT.
SO IT WILL ALSO WORK ON OTHER
THAT WILLATE, AND ALL THE
GLYCANS IT'S VERY--HAVE YOU A
LITTLE BIT OVER OXIDATION, SOME
SIDE PRODUCT CHSES I WILL
ADDRESS LITTLE BIT BUT OVER ALL
IT HAS A VERY SIMILAR PROTILE BY
BLEACH OR BY UPON
[INDISCERNIBLE].
SO THE HPOC CONJUGATION,
FLUORESCENT CONJUSTICE
ACCESSICATION SHOWED THE MAJOR
GLYCANS ARE RELEASED THE SAME BY
BLEACH AND THE PGSF, THIS IS NOT
AS CLEAN AS PGSF, BUT THAT'S
SORT OF EXPECTED AND THE RELEASE
OF THOSE GLYCANS ARE SPHRUCTURAL
INCREASE ARE REALLY VERY WELL
RESERVED SO IF WE PRINT A
MICROARRAY, THE BINDING OF THE
PROTEINS THEY ARE EXPECTED TO,
SO ... THE GOOD THING ABOUT
THESE IS THAT NOW WE CAN WORK
WITH LARGER SCALE TO REALLY
TARGET SOME HIGH MILLIGRAM
COASTAL GRAM SCALE GLYCAN
SEPARATION, THIS IS AN EXAMPLE
THAT WE PURIFY GLYCANS AND THE
GLYCANS LIKE THESE ON THEIR
CURRENT MARKET, YOU'RE LOOKING
AT LIKE A--LIKE 10 TO WENT
MICROGRAM WITH SEVERAL HUNDRED
DOLLARS PRICE THAT IS REALLY
PREVENTING A LOT OF PEOPLE THAT
GET INVOLVE WIDE STUDYING THEIR
FUNCTIONS.
SO AND ALSO WITH THESE AMOUNT OF
MATERIAL, WE CAN GET A VERY NICE
NMR DATA, BOTH PROTON MR AND
CARBON 13 MR, SO FROM ABOUT A
KILOGRAM OF SOY PROTEIN ISOLATES
AND THIS IS APPROXIMATE
SOMETHING PEOPLE TAKE AS A FULL
SUPPLEMENT AND YOU CAN GET THE
LIKE A HUNDRED MILLIGRAMS OF
DIFFERENT STRUCTURE AND IF YOU
NOTICE THERE ARE MONODPLIEWTEN
STRUCTURES THAT LOSE IN THE
TERMINAL GLUNAC, SO THIS IS
INTACT THIS, IS GLYCANS RELEASED
BY BLEACH BUT WE ALSO SEE SOME
OTHER FOR THE TRIAL, YOU CAN
ACTUALLY TRANSFORM THIS
APPROXIMATE BACK TO REDUCING
GLYCANS BY CREATING CARBON.
ALSO ANOTHER VERY INTERESTING
SIDE PRODUCT IS THIS 1 THAT IS
LOSING THE TERMINAL GLUNAC IS A
GOOD SUBSTRATE FOR THE MEDIATED
AND FLIEK O PEPTIDE SYNTHESIS.
WHETHER THIS CAN DO A RELEASE
FOR GLYCO CONJUGATES AND WORK ON
OGLYCAN SHOWN HERE AND THE
IMPORTANT THING IS THE REDUCING
THESE A LITTLE BIT DIFFERENT.
SO THE THESE ARE STABLE TOWARDS
BLEACH.
SO YOU OX DIET THE PROTEIN ALL
THE WAY TO THESE ASSAY, AND THEN
AS STRONG AS YOU THINK PLEACH
MIGHT BE HARSH TO THE
STRUCTURES, IT ACTUALLY VERY
MILD UNDER THIS CONDITIONS,
BECAUSE EVEN [INDISCERNIBLE]
GROUP ON ACID GOT PRESERVED VERY
WELL IF YOU TRADED WITH LIMITED
AMOUNT OF BLEACH.
SO, THIS IS EXPWRUOF THE
DEMONSTRATION OF WORKING ON
O-GLYCANS FROM [INDISCERNIBLE].
SO A LOT OF THESE HAVE LIPID
SAYS AND UNFORTUNATELY THIS IS
ALSO WORKED ONLET PRODUCT AGAIN
IS LIPID, AND IS STABLE, SO THIS
PLEACH OXIDIZE THE WHOLE SERIES
POINTS MID PART AND THIS IS SOME
DEMONSTRATION THAT WILL COME,
AND CAN BE SIZED.
THE GOOD THING ABOUT THIS IS
THAT IT'S NOT ONLY PURIFIED,
THIS BUT THIS WORKED DIRECTLY ON
[INDISCERNIBLE] AND THEN YOU
NEED TO ISOLATE THEM FROM THE
MIX TOUR AND TREATED CHEMICALLY,
GLYCANS ARE VERY DIFFERENT FROM
OTHER BOWEL MOLECULES, AND SO
IT'S--YOU CAN FIND DIFFERENT
WAYS TO SEPARATE THEM.
SO AS A SUMMARY, WE WERE ABLE TO
USE THE SINGLE BLEACH OR
REAGENT, UNDER VERY MILD
CONDITION TO REDEC THESE 3
CLASSES OF DPLI KAN--KANAS.
AND THE MECHANISM, I WOULD SAY
MECHANISM IS PROBABLY MORE
COMPLICATED THAN I SHOW HERE,
AND AS WE SEE A DIFFERENT AS WE
SEE A MORE SUBTLE SIDE,
HOPEFULLY WE WILL REPORT LATER
BUT WE WERE THINKING IT MOSTLY
PROBABLY FROM THIS CLARINATION
INITIATED DEGRADATION IMMUNATION
ON REACTION.
SO JUST THE CHALLENGES COMPARED
TO PREVIOUS METHODS, SO IT'S A
CHEAP REGIONS, SIMPLE OPERATION
AND DIRECTLY APICKABLE TO ANIMAL
PLANT TISSUES INTO HOMOGINIZE
IT, YOU DON'T NEED PREEXTRACTION
OF GLYCO CONJUSTICE ACCESS GAYS,
AND THAT IS THE LARGE SCALE, IS
POSSIBLE.
SO WE ARE WORKING WITH KILOGRAM
STUFF AND ALL YOU NEED IS THE
OVERHEAD AND THE MECHANICAL
STIRRER AND THAT IS THE REACTION
MIXTURE, IT IS AN AQUEOUS
SOLUTION, AND THE SOLUTION GETS
WARM A LITTLE BIT BUT IT'S
NOTHING TO INTERESTING LIKE
[INDISCERNIBLE] THE SCALE THAT
WE TRIED, A HUNDRED LITERS SO
THE DISADVANTAGE, SO THE
CHALLENGE, IT'S NOT AS CLEAN AS
ENZYMATIC CLEAVAGE BUT THIS
STAGE IS MOSTLY USED FOR
PREPARED SCALE AND REACTIONS AND
MEASUREMENTS, I THINK WE ARE
GETTING CLOSER AND CLOSER TO
UNDERSTAND THE OLD MECHANISMS
THROUGH THE ISOLATION OF THESE
SITE PRODUCT BUT WE ARE STILL
WORKING ON IT AND THERE'S
OVEROXIDATION SIDE PRODUCT BUT
SOME OF THEM COULD BE VERY
USEFUL FOR SOME DIFFERENT
OPERATING GLOBALLYLICATIONS,
THIS IS DIFFICULT BECAUSE THERE
ARE MIXTURES TOO REQUIRE
SEPARATION.
WE HAVE MADE SIGNIFICANT
ADVANCES OVER HERE BUT I'M NOW
GOING TO TALK ABOUT THAT TODAY
FOR TIME'S SAKE.
SO IN SORT WE WERE ABLE TO LIKE
BRING THE ISOLATION OF NATURAL
TBLI KAN--KANAS FROM NATURAL
MATERIAL TO THE NEXT LEVEL.
AND REALLY IT BECOMES
ALTERNATIVE [INDISCERNIBLE]
TOWARD COMPLEX GLYCANS SO FOR
NOW, IF YOU ARE WORKING ON LIKE
SOME [INDISCERNIBLE] DPLI
KAN--KANAS, TELL BE VERY HARD
FOR YOU WANT TO DO A SYNTHESIS
EXIST AT THIS POINT.
BUT NOT FOR THOSE MINOR GLYCANS.
--WE HAD PUBLISHED A LITTLE
PAPER THAT USING NBS AGAIN, WE
CAN REGENERATE THE REDUCING
GLYCANS BUT THIS 1 LOOSING A
CARBON IS THE MAJOR SIDE
PRODUCT.
SO MOST RECENTLY, WE ARE WORKING
ON USING DIFFERENT REGIONS,
USING OXOM, WHICH GIVE US MUCH
LESS OF THE PENTOSE, FOR
REDUCING GLYCANS BUT WE LIST
SIDE PART, THIS CAN CONVERT BACK
TO REDUCING [INDISCERNIBLE] BY
REDUCING CARBON.
SO WITH THIS I WOULD LIKE TO
THANK EVERYONE, ESPECIALLY THANK
THE COMMUNITY, THANK PAM, FOR
PUTTING THIS TOGETHER AND THESE
ARE THE PEOPLE WHO DO THE WORK.
THANK YOU VERY MUCH.
[ APPLAUSE ]
>> THANK YOU VERY MUCH.
MY LAB DEVELOPED THEOXIDATIVE
PROCESS FOR THE FIRST YEAR,
UNDERGRADUATE LAB CLASS AND WE
JUST PUBLISHED THAT, AND SO IT'S
STRAIGHT FORWARD ENOUGH THAT
FRESHMAN CAN DO IT.
SO OUR NEXT TALK IS VIRTUAL AND
UNFORTUNATELY DR. DAVID SMITH
COULD NOT BE WITH US HERE TODAY
LIVE AND WE WILL HEAR FROM HIM
VIRTUALLY.
FIRST OF ALL I WANT TO THANK
PAM, AND ALL THE ORGANIZERS OF
THIS MEETING TO PROVIDE THEM A
BRIEF SUMMARY OF THE TECHNOLOGY
THAT WAS DEVELOPED AND YOU KNOW
THIS WAS DEVELOPED WITH MYSELF
AND [INDISCERNIBLE].
NATURAL GLYCANS REAGENTS AND
STANDARDS PREUSING ORNG, AND WE
JUST HEARD SOME DETAILS OF THIS
BUT OUR MESSAGE TODAY FOR
NGLYCAN, OF NATURAL NGLYCANS ARE
AVAILABLE AND AFFORDABLE THANKS
TO THE NIH COMMON FUND AND ALL
YOU FOLKS INVOLVED IN MAKING
THIS HAPPEN.
IN HIS PRESENTATION, DR. SONG
POINTED TO THE FACT THAT LACK OF
LARGE GLYCAN LIBRARIES AND
MILLIGRAM TO GRAM QUANTITIES
LIMITS THEIR USE AS DRUG
TARGETS.
THIS IS TO SUPPORT THIS, I WANT
TO USE OLIGO SACCHARIDES AS AN
EXAMPLE.
HMO IS AN ATTAINABLE FLUID WHICH
IS RICH IN COMPLEX OLIGO
SACCHARIDES, THESE WERE
INTENSELY STUDIED IN THE 1940S
AFTER IT WAS DETERMINE THAD MANY
OF THESE OLIGO SACCHARIDE
STRUCTURES SUPPORT THE GROUP OF
DIFFICULT BACTERIA WHICH WAS A
BENEFICIAL BACTERIA IN THE GUT
OF PREOF THE FED INFANTS AND THE
DATA BUILT THE QUANTITIES OF
THESE OLIGO SACCHARIDES
STIMULATED RESEARCH AND METHODS
FOR SEPARATION AND ISOLATION OF
OLIGO SACCHARIDES.
IN THE EPPED IN THE DEFINITION
OF DPLI KAN--KANA STRUCTURE
SINCE YOU COULD PURIFY THEM AND
THEY WERE AVAILABLE IN MILLIGRAM
QUANTITIES AND MANY OF THEM
STRUCTURAL METHODS BACK THEN
REQUIRED MILLIGRAM QUANTITIES TO
DO THIS WORK.
ONCE THEY WERE DEFINED, THAT YOU
WERE USED AS STANDARDS TO
DEVELOP A MASS SPECTROMETRY,
HELPED DEVELOP MASS
SPECTROMETRY, AS GLYCAN FOR
STRUCTURAL ANALYSIS.
NOW, AS THE HMOs STRUCTURES
BEHAIM DEFINED AND AVAILABLE, AS
I SAID IN THE MILLIGRAM, THIS
RESEARCH WAS CORRECTEDDA AT
DEFINING THE FUNCTION WHICH
INCLUDED PREBIOTIC ACTIVITY AND
THEY ALSO SHOWED TO PROVIDE
PROTECTION IN BREAST FAD
INSTANTS FROM VARIOUS VIRUSES
LIKE INFLUENZA AND ROTOVIRUS,
BASED ON THEIR RECEPTOR DECOY
REACTIVITY.
MOST RECENT STYLES HAVE SHOWN
THAT EFFECTS CONTRIBUTE TO
INFANT HEALTH AND IN 2014 A
NUMBER OF INFANT FORMULA
MANUFACTURES INTRODUCED THIS AS
AN ADDITIVE TO THEIR PRODUCT AND
ALL THESE ACCOMPLISHMENTS OF
COURSE WERE POSSIBLE BECAUSE OF
THE BIOLOGICAL GLYCANS WERE
AVAILABLE IN THE LARGE NOWOF THE
QUANTITIES TO DO THE STUDIES.
ANOTHER EXAMPLE OF THE FLUID
RICH IN GLYCAN IS HUMAN OVANIAR
ASSIST FLUID AND IN THE EARLY
DAYS OF MEDICAL CARE, THIS WAS A
FLUID THAT WAS READILY AVAILABLE
AND IT CONTAINED MUSEINS THAT
CARRIED THE ABO BLOOD GROUPS OF
THEIR DONOR.
AND BECAUSE OF THEIR
AVAILABILITY AND LARGE
QUANTITIES, THEY WERE USED AS
THE STRUCTURES FOR DETERMINING
THE ACTUAL SEQUENCE OF THE ABO
AND LEWIS BLOOD WORK SYSTEMS.
SO USING THOSE AS AN EXAMPLE,
NDPLI KAN--KANA WAS FOUNDED ON
THE CONCEPT THAT IF WE COULD
MAKE MAMMALIAN DPLI KAN--KANAS
AS AVAILABLE, AS HMOs, OR AS
OVERYIAN SIS FLUID, WE MIGHT BE
ABLE TO CONTRIBUTE TO ADVANCING
OUR UNDERSTANDING OF THE DPLI
KAN--KANA FUNCTIONS AND THIS
SEEMS TO BE A MAJOR--A MAJOR
GOAL OF THE SYNTHETIC EFFORTS IN
THE COMMON FUND WORK.
WE HAVE A NUMBER OF DIFFICULTS,
WE KNOW THAT SYNTHESIS AND
SEQUENCING OF LARGE AND NATURAL
GLYCAN SYSTEM DIFFICULT, AND AS
MENTIONED, YESTERDAY, UNLIKE
OLIGO NUCLEOTIDES AND PROTEIN
AMPLIFICATION OF PROTEIN
STRUCTURES AND NOT A ROUTINE
PROCESS, ALTHOUGH WE ARE WORKING
ON SOME OF THOSE IDEAS.
AND NATURAL DPLI KAN--KANAS ARE
INDEED IN LOW ABUNDANCE IN
NATURAL PRODUCTS.
SO WHAT WE NEED IS A CHEMICAL
METHOD THAT WE CAN SCALE UP SO
AT NET TPHREU RACING KAN--KANA
WE START WITH LARGE QUANTITIES
OF NATURAL PRODUCTS AND THEN WE
APPLY ORANGE AND SOANCH A
SCALABLE PROCESS AND WE
DETERMINED AT THIS POINT, THAT
THE SCALE OF OF THE PROCESS IS
LARGELY DEPENDENTOT SIZE OF THE
EQUIPMENT AND IF WE CAN
CAPITALIZE ON THIS ECONOMY OF
SCALE AND WE'RE WORKING ON THAT,
IT MAY BE POSSIBLE REALLY TO
SUPPLY MILLIGRAM QUANTITIES OF
GLYCANS AT VERY LOW COSTS.
NOW THANKS TO NIGMS TO DR. SONG
IN THE EARLY DAYS AND PHASE 1
PROJECT, TO NET GLYCAN, WE
DEMONSTRATED THE FEASIBLE OF
THIS APPROACH.
AND AS I'M SHOWING YOU HERE, YOU
SEE THE SLIDE BEFORE, BUT THIS
JUST SHOWS THE ORANGE PROCESS AS
IT--CAN REMOVE DPLI KAN--KANAS
FROM ANY GLYCO CONJUGATE, BUT WE
FOCUSED ONURE TECH ON NGLYCANS.
NOW AFTER DEMONSTRATING THESE
ABILITY, WE WERE AWARDED A PHASE
2 GRANT WHICH IS NOW SUPPORTING
THE NRM PRODUCTION OF DPLRKS
DPLI KAN--KANAS AND WE
ACCOMPLISHED THIS BY
THEOXIDATING PROCESS TO CONTROL
BYPRODUCTS BY PREPARING BI
FUNCTIONAL TAGS SO YOU FOLLOW
THE PUREUNCTION AND THEN THESE
CAN BE IMMOBILIZED FOR A VARIETY
OF FUNCTIONAL STUDIES AND WE'VE
DEVELOPED REMOVABLE TAGS TO
FOLLOW PURIFICATION AND THEN,
REMOVE THEM SO THAT WE CAN HAVE
ACCESS TO THE FREE NATURAL
GLYCANS.
AND WE'VE DEVELOPED
PREVENTIVERATIVE PURIFICATION
METHODS TO PURIFY DPLI
KAN--KANAS, AND THIS
PURIFICATION PROCESS AS EVERYONE
TOLD US EARLY ON WAS THE IMPABL
PART IT WAS DIFFICULT AND WE'RE
STILL WORKING ON IT BUT IT IS
PROGRESSING AS I SAID WE FOCUSED
OUR FIRST ACTIVITYOT HIGH
MANNERS IN DPLI KAN--KANAS AND
WE'RE HOW HAPPY TO REPORT THAT
WE HAVE OVER A GRAM OF MAN 9 IN
INVENTORY AND A HUNDRED
MILLIGRAM QUANTITIES OF THE
MAJOR ISOMERS OF THE STRUCTURES,
NOW THE MINORS THERE, ARE
CERTAINLY THERE BUT IN SMALLER
QUANTITIES BUT THEY ARE THERE
THESE DPLI KAN--KANAS ARE
AVAILABLE IN A VARIETY OF
DERIVATIVES THAT CAN BE USED FOR
STANDARDS AND MARKERS WE HAVE
THEM WITH FUNCTIONAL GROUPS THAT
CAN BE ATTACHED TO VARIOUS
IMMOBILIZED CERTAINTIES AND WE
CAN MAKE THEM WITH BIOTIN AS
WELL SO THESE ARE READILY
AVAILABLE FOR ANALYSIS, SO THIS
MEANS THAT WE CAN NOW SAY THAT
WE HAVE LARGE QUANTITIES OF PURE
NATURAL GLYCANS AVAILABLE AS
NEEDED TO DEFINE GLYCAN
STRUCTURE.
NOW THE CHALLENGE TO OUR
COMMUNITY, I BELIEVE NOW IS WHAT
DO WE DO WITH THESE OLIGO
SACCHARIDES.
OUR HOPE IS THAT FOLKS WILL WANT
TO PURCHASE THEM AND USE THEM IN
THE EXPERIMENTS FOR GLUE MARIOUS
KOAMIC STUDIES AND SOME OF THOSE
STUDIES MIGHT INCLUDE THINGS
I'VE LISTED HERE, WE NOW REALLY
HAVE UNLIMITED QUANTITIES THAT
CAN BE USED FOR STAND ARPDS FOR
METHODS OF DEVELOPMENT AND
DEFINING STRUCTURES.
SINCE WE HAVE--WE CAN MAKE
LARGER AMOUNTS, WE CAN USE THESE
AS PRECURSOR FOR ELONGATION OF
OTHER STRUCTURE BY ENZYMATIC
METHODS, AS DR. SONG MENTIONED
THE SUBSTRATES FOR THE MUSEUM
STATED END-TO-END IS A MAJOR
BYPRODUCT IS A MAJOR DERIVATIVE
AND DR. LEE'S GOING TO TALK
ABOUT THAT IN A LITTLE WHILE.
CO CRYSTALLIZE [INDISCERNIBLE]
CAN BE USED FOR ISOLATION FOR
PULL DOWN EXPERIMENTS TO PULL
DOWN SPECIFIC GLYCAN BIND
PROGTEENS, GLYCAN CONJUGATES CAN
BE USED TO SCREEN FOR ANTIGLYCAN
REAGENTS BY PHASIA'S PLAY AND
OTHER EFFORTS AND DR. CUMMINGS
HAS DONE A LOT OF WORK IN THAT
AREA NADDITION TO CELLULAR
RESPONSES THAT DEFINE FUNCTION
FUNCTIONS OF POSSIBILITY AND
EVEN ANIMAL STUDIES ON SMALL
ANIMALS FOR LOOKING AT POTENTIAL
SYSTEMIC EFFECTS OF NGLYCANS OR
OTHERWISE ADMINISTERING DPLI
KAN--KANAS CAN BE POSSIBLE FOR
WE'RE LOOKING FOR PEOPLE TO
BEGIN APPROACHING AND THINKING
OUTSIDE THE BOX ABOUT OTHER
METHODS THAT CAN BE USED TO DO
THIS AND WE HAVE A NUMBER OF
GLYCANS NOW, IN OUR INVENTORY.
IRWANT TO BREFLY GO THROUGH
THESE, THESE ARE THE COMPLEX
BINARY TPHREU RACING KAN--KANAS
WE HAVE THEM IN PRIMARILY IN THE
SIXTH SIGNIFIAALATED
[INDISCERNIBLE].
THESE ARE HUNDRED MILLIGRAM
QUANTITIES.
WE HAVE THE DERIVATIVES OF ALL
OF THESE STRUCTURES.
WE'VE ISOLATED A NUMBER OF
HYBRID STRUCTURES.
THESE ARE SOME OF THE MAJOR 1S
WE HAVE, THERE ARE A LOT MORE
COMES ALONG, AND IT'S COMING
SOON, ALSO WILL BE THE
MULTIITENIARY GLYCANS THAT ARE
WORKING ON THOSE.
SO IN SUMMARY, I WANT TO JUST
SAY THAT NET DPLI KAN--KANA
SPECIALIZING IN NATURAL
GIVING--YOU KAN--KANAS FOR
FUNCTIONAL GLYCANS.
WE HAVE NDPLI KAN--KANAS AND
NGRAM QUANTITIES WITH PURITYS
GREATER THAN 90%.
OGLYCANS AND NGLYCANS ARE COMING
IN THE FUTURE AND WE'RE PREPARED
TO WORK WITH ANYBODY WHO WANTS
TO FIND THE DPLI KAN--KANA OUT
THERE.
WE FEEL LIKE THEY'RE THERE, AND
AND WE SHOULD BE ABLE TO
IDENTIFY THEM FOR YOU AND WE
FEEL LIKE THIS IS IN LINE WITH
FEDERAL GRANTS.
WITH THAT I WANT TO SAY THANK
YOU FOR YOUR TIME AND WE LOOK
FORWARD TO YOUR ORDERS.
[ APPLAUSE ]
EMPLOY.
>> THANK YOU SO MUCH DR. SMITH.
AND CONTINUING IN OUR THEME OF
BASIC SCIENCE TRANSLATED TO
COMPANIES OR OTHER BASIC BIOLOGY
STUDIES, I INTRODUCED
DR. PRUDDEN AND BOONES.
--SERIES OF DPLI COSDACE AND
TRANSFER ACES TO CONSTRUCT A
NUMBER OF IMPORTANT SUBCLASSES
OF NGLYCANS BEING
[INDISCERNIBLE] STRUCTURES
COMPLEX AND HYBRID STRUCTURES.
THE TALK TODAY WILL FOCUS ON THE
COMPLEX AMONG THEMSELVES.
SO THERE ARE A NUMBER OF
IMPORTANCES THAT HAVE BEEN
OBSERVED WITH NDPLI KAN--KANAS 1
OF THEM BEING THE MOST IT IS 1
OF THE MOST COMMON AND COMPLEX
POST TRANSLATIONAL
MODIFICATIONS.
THEY'RE RELEVANT TO A NUMBER OF
BIOLOGICAL PROCESSES, AND
GLYCOSYLATION OF ANTIBODIES HAS
BEEN SHOWN TO MEDIATE CELL
CYTOTOXICITY.
WE ALSO KNOW THAT CHANGES IN
DPLI COSALATION OF PROFILE CAN
CHANGE DURING DISEASE STATES
WHICH MAKES DPLI COSALATION AN
INTERESTING TARGET FOR BIOMARKER
DEVELOPMENTS.
ALTHOUGH THE IMPORTANCES ARE
WELL UNDERSTOOD, THERE ARE A
NUMBER OF CHALLENGES, SOME OF
WHICH WE'VE ALREADY HEARD,
HETEROGENERATED AITY, OF N-DPLI
KAN--KANA CANS DEPEND ON CELL
TYPE AND MICRO ENVIRONMENT.
THESE STRUCTURES ARE ISOBERNEA
DEETIC, MEANING THAT THEY CAN
HAVE THE SAME MONOSACCHARIDE
CONNECTIVITY, OR MONOSACCHARIDE
PROFILE, WITH DIFFERENT
CONNECTIVITY GIVING THEM
IDENTICAL MOLECULAR WEIGHTS.
IT CAN BE ALTHOUGH THERE HAVE
BEEN IMPROVEMENTS DIFFICULTY IN
ASSESSING INDIVIDUAL WELL
DEFINED STRUCTURES FROM NATURE
AND SUCH ISOLATION DEPENDS ON
[INDISCERNIBLE], ADDITIONAL
SOURCES ARE COMING ON ONLINE AND
THE GOAL OF OUR NIH SBIR IS TO
UTILIZE THE ENZYMATIC
METHODOLOGY FOR SIPGHTICIZING
THE 20 MOST ABUPPED ANT GLYCANS
DONE IN HUMAN SERUM, ALL OF
WHICH HAVE A GREAT--HAVE AN
ABUNDANCE GREATER THAN 1%,
TOGETHER THAT MAKES UP 50% FOR
THE TOTAL SERUM NDPLI KOAM,
UNFORTUNATELY A SIGNIFICANT
POPULATION OF PROTEINS WHICH
INCLUDE PROTEINS THAT ARE
GLYCOSYLATED, THE LIBRARY WE ARE
60ICIZEING AS INDIVIDUAL
STRUCTURES WILL CONTAIN A FREE
REDUCING TERMINUS, REDUCTIVE
AMDISMAGZ THIS ALLOWS FOR
EXTREME FLEXIBILITY AND USER
WHETHER IT'S LCMS, MSMSOR OR CAP
ILLEGALSARY ELECTROFORRIESIES,
THE PRODUCT IS AMENABLE TO SUCH
APPLICATIONS, ADDITIONALLY THESE
ARE PURIFIED BY LCMS AND THEIR
STRUCTURES ARE CONFIRMED BY NMR,
AND MOST IMPORTANTLY THE LIBRARY
TO BE SYNTHESIZED WILL CONTAIN A
UNIFORMLY CLICK NAC WHICH CAN BE
USED ANAIN ERNE TERNAL
[INDISCERNIBLE].
MAKING THIS PRODUCT CAPABLE OF
DETERMINING DPLI KAN--KANA
CONCENTRATION CHANGES WITH
REGARDS TO DISEASE PROGRESSIONS,
THIS WILL BE IMPORTANT FOR THE
BIOMARKER SECTOR.
AS YOU CAN SEE, THEY ARE VERY
DIVERSE, THERE ARE
NATURAL--NEUTRAL STRUCTURES, WE
HAVE A, SIDIC STRUCTURES BEARING
1 OR 2 SALIC ACIDS, THESE
SIALATED CAN BE ASYMMETRIC WHERE
WE HAVE DIFFERENT 1S IN EACH
ARM, [INDISCERNIBLE] GLUE
COSALATED AND ALSO STRUCTURES
WITH ISOMERIC SIALICK ACID
PLACEMENT ALTHOUGH WE HAVE SENT
OUT THE TARGETS, THE PROTOCOL WE
USE IN THE NEXT SLIDE OR 2, CAN
PROVIDE ACCESS TO OTHER ADVANCED
INTERMEDIATE THAT WILL BE USEFUL
STANDARDS, SO OUR STRATEGY
RELIES ON AN ADVANCED STARTING
MATERIAL THAT WE EXTRACT FROM
THE EGG YOLK POWDER, THAT
EXTRACTED MATERIAL IS DIGESTED
TO FOR THE [INDISCERNIBLE]
SACCHARIDE WHICH IS COMMON TO
ALL NDPLI KAN--KANAS.
OR ALL HUMAN NGLAI KAN--KANAS.
AND VERY BRIEFLY, THIS IS HOW WE
INSTALL OUR C13 LABELED TAG.
STARTING FROM THE CORE
APENTOSACCHARIDE, AND USE GLYNAC
ON INSTALL THE ARM TO CREATE THE
DESIRED HEXASACCHARIDE.
THIS IS HEXASACCHARIDE IS FOR
MDN2, AND WE CAN USE THE CARBON
13 TO SELECT THE INSTALL 1, C13
LABELED GLYCNAC ON THE BRANCH
TREATING THE SACCHARIDE.
THIS HELPED THE SACCHARIDE THEN
CAN BE EXTENDED IT COULD BE
EXTENDED USING TRANSFER ACES, TO
CREATE 2 LAC INCOME AY, AND THEN
DOZE CAN BE MODIFIED WITH
VARIOUS SILO TRANSFERAISES TO
CREATE THE DESIRED TARGETS.
IMPORTANTLY AS CAN YOU SEE FROM
THE SCREAM ISSUES NOT ONLY DO WE
GET THE DESIRED DISCIPLINARY
SOLID STRUCTURES, BUT ALSO
INTERMEDIATE PROVIDE TARGETS OF
THEIR OWN IMPORTANCE.
FURTHER MORE THE INSTALLATION OF
A UNIFORMLY INSTALLED GLYCNAC AT
A PARTICULAR SITE DOES ALLOW FOR
FRAG M-TEBURKEULOSEISATION
ANALYSIS WHICH IS MORE DIFFICULT
ON A STRUCTURE THAT IS NOT C13
LABELED.
AS I MENTIONED IN THE PAST, WE
LOOK TO MAKE THESE PURE AND WE
DO THAT BY USING CHROMOING TO
RAFALLIC TECHNIQUES, I
ILLUSTRATED 1 EXAMPLE HERE WHERE
WE CAN INSTALL THE CORE FUCOS,
AND USING HELIOS POSITIVIC LCMS,
WE WERE ABLE TO SUG95 CAN'TLY
DIFFERENTIATE OUR DESIRED
PRODUCT FROM ANY UNREACTED SUGAR
NUCLEOTIDES FOR REACTION
COMPONENTS.
THIS HOW WE'RE ABLE TO GET HIGH
PURITY STRUCTURES, WITH ONLY 1
TARGET SO THERE WILL BE NO
MIXTURES IN THESE PRODUCTS.
WE ARE LOOKING AT A VAST LIBRARY
TO LOOK AT STANDARDS ISSUES THE
STARTING MATERIAL THAT WE BEBIN
WITH IS EXTRACTED FROM EGG YOLK
POWDER AND WE USE RECOMBIN ANT
STRANS FER ACES TO YIELD
ISOMERS, WE USE AN UNNATURAL
GLYCNAC TO ASYMMETRIC TARGETS
WHICH I DON'T HAVE TIME TO
DISCUSS HERE BUT THIS IS
AVAILABLE ON A PAPER WE PUBLISH
INDEED 2019.
HPLC PURIFICATION RESULTS IN
ANALYTICALLY PURE MATERIAL, I
WANT TO STRESS THESE HAVE FREE
REDUCING--PREE REDUCING TARGETS
SO THEY'RE AMENABLE TO A NUMBER
OF APPLICATIONS.
COMPREHENSIVE INCLUSION OF
UNIFORMLY LABELED CARBON 13
REPRESENTS A UNIQUE COLLECTION
OF TARGETS THAT COMPRISE WITH
50% OF THE SERUM DPLI KOAM AND
WE CURRENTLY ARE IN DISCUSSIONS,
COMMERCIAL PARTNERING WITH
DISCUSSIONS AS THEY HAVE AN
INTEREST IN WORKING WITH US ON
THESE CARBON 13 LABELED TARGETS.
SO WITH THAT JUST A COUPLE OF
ACKNOWLEDGMENTS OF COURSE THIS
FUNDING WAS PROVIDED BY THE NIH
THROUGH A CURRENT SBIR PHASE 1
AND THIS--THIS TECHNOLOGY WAS
ACTUALLY STARTED DURING MY
Ph.D. WORK WHICH WAS FUNDED BY
A PREDOCTORAL FELLOWSHIP SO I
HAVE TO GIVE ANOTHER HATS OFF TO
NIH AS WELL, OF COURSE THE
COMPLEX CARBOHYDRATE CENTER AND
WE HAVE 2 WEB SITES THAT ARE
UNDER CONSTRUCTION.
THIS WORK IS BEING DONE OF
COURSE ALSO WITH
DR. [INDISCERNIBLE], AND
DR. [INDISCERNIBLE].
IF ANYONE HAS ANY FURTHER
QUESTIONS CONTACT US WITH E-MAIL
AND I APPRECIATE THE TIME TO
SPEAK TODAY.
>> MANY THANKS.
[ APPLAUSE ]
OUR NEXT SPEAKER IS LIVE WITH US
HERE IN BETHESDA, DR. LI FROM
GEORGIA STATE UNIVERSITY.
>> FIRST OF ALL I WOULD LIKE TO
EXTEND COLLEAGUES AT THE NE H
ORGANIZE THIS WONDERFUL MEETING
AND IT'S REALLY A GREAT
ALTERNATIVE FOR US TO MEET
TOGETHER, ESPLEE FOR ME AS A
JUNIOR FACULTY TO MEET ALL THE
OTHERS TO TALK WITH THEM, TO I
WILL TALK AND OGLYCANS, I HAVE A
POINTER.
SO I WILL START WITH THIS
[INDISCERNIBLE] I WANT TO SHOW
THIS BEFORE, SO, POINTER--OH,
OKAY GO, BACK, SO, OKAY, SO
NEVER MIND, AS CAN YOU SEE, OVER
HERE, THE--SO THE GLYCANS,
OGLYCANS ACTUALLY ACTUALLY IS
PRETTY ABUNDANTOT SURFACE, SO WE
BASICALLY FOCUS ON THE GLYCANS
OR ON THIS PROJECT--APPLY THESE
STRUCTURES, USE THEM AS
STANDARDS OF PROBES OR USE THAT
AS BIOMARKERS SO THIS IS A
SUCCESSFUL STORY ABOUT THIS
WHICH IS A GLYCO PROTEIN AND USE
THAT AS VACCINE DEVELOPMENT AND
THERAPEUTICS FOR EXAMPLE, THESE
CAN BE USED FOR DISEASES.
--BUT CAN CAN BE REALLY
COMLICATED HERE, BUT WE LOOK AT
THIS IN DETAIL, THERE ARE OTHER
COURSE GLYCANS AND SOME OTHER
MINOR STRUCTURES OVER HERE, SO
THEY'RE REALLY LOW ABUNDANCE.
I MEAN DOUG TOLD YOU HOW TO
SEPARATE THESE BUT FOR THE LOW
ABUNDANCE WE THINK THESE WILL BE
THE BETTER CHOICE, SO OF COURSE
WE LOOK AT THESE CORE
STRUCTURES,----SO, OF COURSE,
FOR THE OLD SELECTING THE SAME
THING, YOU HAVE THE COURSE, YOU
HAVE MULTIPLE EPIEPITOPES, SO
HERE IS BASICALLY IS IN-BOUND
FOR THE USE HERE, THIS IS A LOT
OF GLYCANS ON THE GLYCANS HERE.
FOR THIS.
AND OUR PROTEIN HERE IS
OBVIOUSLY WE TRY TO USE CHEMICAL
WAYS TO SYNTHESIZE THE REALLY
CONSERVED CALL STRUCTURES, FOR
EXAMPLE, WE HAVE THIS BEFORE,
AND RIGHT NOTICE WE BASICALLY
APPLY THESE METHODS OF COULD YOU
SAYS, TO ODPLI KAN--KANAS.
ONE OR 2 OR 3 SUGARS WHICH ARE
RELATIVELY EASY TO SYNTHESIZE IN
LARGE SCALE BUT CAN YOU SEE THE
PROTEINS, AND THEN WE HAVE ALL
THESE MATERIALS OR ENZYMES TO
[INDISCERNIBLE].
AND LATER ON, WE WILL TRY TO USE
THE SENSES TO SIMPLIFY SENSES.
I WOULD JUST USE 1 SLIDE TO
SUMMARIZE WHAT WE HAVE DONE NOW
BASICALLY WE STARTED WITH THIS,
WE DESIGNED THIS REALLY
UNIVERSAL BUILDING BLOCK OVER
HERE AND THEN WITH 2, THE FIRST
STEPS WE CAN BASICALLY CONVERT
THIS 1 TO ALL THE COURSE, A
COURSE AND YIELDS FROM 51%, TO
84%.
SO THESE ARE ALL FOR THE SYNTH
ASSESSING SOLID BASE, AND THEN,
WE CAN ALSO PROTECT THAT FOR THE
TPHREU RACING KAN--KANA SYNTHASE
FOR THE WILD 2 OR 3 STEPS,
USUALLY THE YIELD IS PRETTY
GOOD.
I WILL SKIP THIS, IT'S THE SAME
THING--I LITTLE BIT UP SO WE
HAVE ALL THESE STANDARD
OPERATIONAL PROCEDURES
AVAILABLE, SO WE HAVE PUT ALL
THIS GLYCO CULTURES AND THESE
CRITIC STEPS SO PEOPLE WANT TO
REPRODUCE, TO REPEAT IT, IS THEY
CAN REALLY FOLLOW THESE
PROCEDURES IN THIS WEBSITE AND
ALSO MY OWN GROUP WEBSITE.
SO WE HAVE ENZYMES AND THIS
ENZYMES AND EXTEND THESE TO THE
NGLYCANS AND WE PEPTIDES AND
ALSO ALSO THIS IS PRETTY
EFFICIENT, SO WE COLLECTED
ENZYMES AND LATER ON WE TRY TO
ABOUT 5 OR 6--SO THIS AS 2
BRANCHES IT BRANCH CAN EXTEND ON
THE GLUNAC AND THIS CAN EXTEND
ONED [INDISCERNIBLE].
SO WE CAN TEST THESE USING VERY
SIMLE IF GIVES SOME ROBUST TOOLS
TO REALLY RAPIDLY EXTEND MAYBE 1
OR 2 CORE STRUCTURES CAN
SYNTHESIZED CHEMICAL TO MAYBE
DOZENS OR EVEN 10S OF
STRUCTURES, I WILL GIVE YOU 1
EXAMPLE HERE, SO, THE SENSES ARE
DPLI KAN--KANAS SO AGAIN
STARTING WITH THE BLOG ISSUING
WE SYNTHESIZED A FEW CLAUSE
CHEMICALLY AND WE COMBINE THIS
WITH ANOTHER 13 ENZYME ADIOS AND
IN THESE AUDYES WE WILL
HAVE--EACH 1 WILL HAVE A
SPECIFIC ENZYME WHICH CAN
TRANSFER AND RECOGNIZE SINGLE
ACCEPTABLE AND CAN CATALYZE THE
SYNTHESIS OF A SINGLE GLYCO
SITTIC BOND.
SO THESE WE ALSO HAVE SUGAR
NUCLEOTIDES AND I THINK THEY
WILL TALK ABOUT THESE, THE
COMMERCIALIZE THESE THEY ARE
PRODUCE THESE IN REALLY LOW
PRICES.
SO 1 EXAMPLE HERE TO EXTEND THE
CORE TO, SO I MEAN FROM 1 WE CAN
DESIGN AND THIS CAN BE FURTHER
EXTENDING FOR SURE, UP HERE.
SO YOU CAN REALLY GET A LOT OF
STRUCTURES.
BUT AGAIN, SO [INDISCERNIBLE]
BASED ON THE SPECIFICITY OF THE
ENZYMES AND FOR EXAMPLE,--FOR
THIS PAPER, WE SYNTHESIZED ABOUT
80 OF THESE GLYCANS AND OF
COURSE, FOR [INDISCERNIBLE]
GLYCANS WE CAN START IT FROM
BLOCKS HERE AND TO SYNTHESIZE
MAYBE 30 OR MORE STRUCTURES OVER
HERE.
SO WE USE THESE TO DO A
MICROARRAY AND TEST ACTIVITY BUT
I WILL NOW SHOW HERE.
AND WITH THAT, EVEN THOUGH THE
ENZYMES AREENTIOUS FICIENT, I
STILL NEED SOME EXPERTISE TO
DEAL WITH THIS AND THERE'S A LOT
THOSE THAT CAN TAKE EFFORTS, I
THINK OUR GOAL AND THE GOAL OF
THIS COMMUNITY, OF THESE COMMON
FUND PROGRAM IS TRY TO SIMPLIFY
THESE AND MAYBE IDEALLY TO DO
AUTOMATIC SENSES, I THINK YOU
LEARN FROM [INDISCERNIBLE] VERY
SOON ABOUT THE CHEMICAL
SYNTHESIS WHICH USES AUTOMATIC
[INDISCERNIBLE].
SO WHEAT TRYING TO COMBINE THIS
ENZYMATIC STUFF INTO THIS
AUTOMATIC SYNTHESIS.
SO WHAT WE DO IS WE USE THE
SYNTHESIZER, WE PUT ALL THE
REGIONS IN THE TUBES AND
ENZYMES, NUCLEOTIDES AND OTHER
IONS AND THEN WE CONJUGATED THE
STARTING GLYCAN ON TO A RISEN OR
SOMETHING INTO THIS VILE AND CAN
PROGRAM IT TO DO THE SIPGHT SIS.
[INDISCERNIBLE] LEADED THIS
PROJECT, THIS 1.
SO THE PROBLEM IS SO THE PROBLEM
IS THESE ARE NOT SOLID FACE, THE
SOLIDS WE USED FOR THE 60 SIS,
IT HAS A LITTLE [INDISCERNIBLE]
SO IT CAN INCREASE LOW INCAP
SIDITYY BUT ENZYMES LOOKS LIKE
THEY'RE NOT SO GOOD AT GOING
INTO THE [INDISCERNIBLE] BECAUSE
THEY'RE RELATIVELY LARGE, SO THE
EFFICIENCIES, WHAT WE DO OVER
THERE IS WE USE THE MATERIAL
HERE, SO AT LOW TEMPERATURE
WHICH ENZYME WILL WORK, OR
30-DEGREE TO THE SYNTHESIS AND
THEN IT WILL PRESS IT OUT.
AND THIS--THIS SYNTHESIZER, SO
WE HAVE 3 STEPS SO WE WILL
LOAD--FIRST LOAD THE TPHREU
RACING KAN--KANAS ON TO A RISEN,
ON TO A POLYMER AND THEN WE ADD
ALL THIS HERE, WITH THE PROGRAM
AND THEN WE CAN INCREASE THE
TEMPERATURE TO ROOM TEMPERATURE
OR MAYBE JUST A DEWITH THE
[INDISCERNIBLE].
AND THEN AFTER DIRECTION TELL
INCREASE TEMPERATURE TO
[INDISCERNIBLE] TO
[INDISCERNIBLE] ENZYMES TO
REMOVE ALL THE SUGAR NUCLEOTIDES
OR OTHER STUFF AND WE CAN DO
THIS CYCLE BY CYCLE AND
SYNTHESIZE ALL THESE LITTLE BIT
COMPLEX STRUCTURES.
AND ALSO WHAT WEMENT TO DO IS WE
ALSO WANT TO DO THE GLYCO
PEPTIDE SYNTHESIZE, THIS THING
IS AGAIN, ENZYMES ARE NOT
COMPATIBLE WITH SOLID PHASE, AND
WHEN AGAIN BECAUSE THEY
CANNOT--I MEAN SNEAK INTO THE
SMALL PORES, RIGHT NOW WE CHAIN
THAT INTO AN ACIDIC BOWL.
SO THE CAP SID HERE, SO
BASICALLY WE ONLY HAVE THE DPLI
KAN--KANAS LOADING ON THE
SURFACE OF THE BALL.
THE LOADING CAP SID IS CLEARLY
PRETTY LOW BUT IT'S BETTER FOR
ENZYMES TO WORK, SO LAWN MOWER
WE DO IS BASICALLY WE--WE HAVE 2
SIDES, SO 1 SIDE ON THE LEFT, WE
WILL DO THE SOLID FACE PEPTIDE
SYNTHIZE, EVERYTHING IS SAME TO
THE TRADITIONAL SYNTHESIS, THE
ONLY DIFFERENCE IS WE WILL
CHANGE THIS TO [INDISCERNIBLE],
AND AFTER WE HAVE ALL THESE
OORK, SO WE WILL INTRODUCE THIS
YEAR AND WE WILL GET SOME
PEPTIDES WITH THE
[INDISCERNIBLE].
AND WE WILL TRANSFER THAT INTO
AN AQUEOUS SOLUTION, AND ALL OF
THESE CAN BE [INDISCERNIBLE] AND
NORMALLY WE HAVE THESE, WE HAVE
ALL THESE OTHER STRUCTURES WITH
PEPTIDES WE JUST DO THE PROGRAM
AND THE MACHINE WILL DO ALL THE
STUFF.
SO TO SUMMARIZE, WE HAVE ALL
THESE AVAILABLE ONLINE, HOW TO
CALCULATE THROUGH THE COURSE AND
ENZYMES SO WE TRY TO CLICK INTO
THESE LINKS HERE, WE SEE HOW TO
EXPRESS THESE ENZYMES HOW TO
PURIFY, HOW TO CHOOSE THE ASSAY,
AND WHAT IS SPECIFICITY OF THE
ENZYMES, SO THIS IS REALLY A
DETAILED INFORMATION HERE.
AND OF COURSE, THE VALIDATION,
DO THIS VALIDATION AND WE ALSO
HAVE COLLABORATIONS, SO THEY GOT
OUR CLOTHES AND OUR PEPTIDES
THERE THAT WORK.
[INDISCERNIBLE] WE PUBLISHED
THESE PAPERS.
AND FINALLY, I WILL SKIP ALL
THESE PAPERS, SO FINALLY, I
WOULD LIKE TO THANK DOUG'S GROUP
FOR DOING THE CROSS VALIDATION
AND THE SHARE THE ENZYME AND THE
CYTOTRANSFER DISEASE AND THIS IS
A GROUP OVER HERE AND ALSO MOST
IMPORTANTLY THE GLYCO COMMON
FUND TO SPOIL US AND RIGHT NOW
WE HAVE THIS GROUND TO
SYNTHESIZE PEPTIDES AND WE HAVE
THIS SBIR TOGETHER WITH
[INDISCERNIBLE] TO SYNTHESIZE
MUSEIN PEPTIDES, SO THAT'S ALL.
THANK YOU.
[ APPLAUSE ]
>> THANK YOU.
SO OUR NEXT SPEAKER TODAY IS BY
DR. POLIZZI, FROM CHEMILY.
>> IT'S A PLEASURE TO SPEAK WITH
YOU TODAY.
SO THE PREVIOUS SPEAKER VS BEEN
SO KIND TO INTRODUCE A LOT OF
IMPORTANT SUGARS TO YOU SO
THEY'VE DONE GROUND FOR FOR ME,
AND HOW SHOWS SUGARS ARE AND
WHERE THEY'RE COMING FROM, FOR
TODAY, SO I SPEAKING TO YOU, WHY
CHEMILY, LLC, AND WE ARE
SPEAKING TO YOU ABOUT THESE
REAGENTS AS THE PREVIOUS
SPEAKERS AS WELL, SO I WANT TO
START WITH WITH ACKNOWLEDGMENTS
AND HE IS THE GROUP THAT WAS
UNDER GEORGE WONG AT THE GEORGIA
STATE UNIVERSITY HAVE REALLY
DEVELOPED A LOT OF THE METHODS
AND DONE A LOT OF CLONING AND
ENZYMES ARE GOING TO BE
IMPORTANT FOR WHAT I'M SPEAKING
TO YOU ABOUT RIGHT NOW AND THEN
WE'VE TAKEN SOME OF THEIR GREAT
TALENT FROM THAT LAB, AND
ACTUALLY MOVE THEM OVER INTO
CHEMILY, AND ALL COLLEAGUES HAD
BEEN AT THAT LAB IN THAT STATE.
I ALSO WANT TO THANK THE FUNDING
AS WELL, AND REITERATE FROM
YESTERDAY IT WAS INTERESTING AND
NOVEL FOR THE COMONT COMMON FUND
TO PROVIDE THEIR OWN SBIR AND WE
WERE FUNDED UNDER THE NEW
TECHNOLOGIES FOR THE FLIEK O
SCIENCES SOLICITATION.
SO JUST A LITTLE THOUGHT
EXPERIMENT, SO MAYBE A BIT ABOUT
YOUR EXPERIENCES SO WOULD YOU
LIKE YOUR UNDERGRADS MAKE A GTP
SOLUTION?
MAYBE SO.
KNOWING THEY WILL SPILL SOME,
WE'VE HEARD HEARD FROM NIKI THAT
WE ARE TRYING TO GET THESE INTO
OUR UNDERGRADUATES AND OUR NOVEL
SCIENTISTS HANDS, KNOWING
THEY'RE GOING TO RUN PC R
EXPERIMENTS, IT'S NOT ABOUT
MAKING SLIEWGDZS, IT'S ABOUT
WHAT YOU WILL DO WITH WITH THEM
AND OVER AND ORDER AGAIN AND
DEVELOP THE SKILLS AND WE SWITCH
THAT OVER TO FLIEK O EXPNS AND
THIS MAKES YOU MORE NERVOUS,
WILL YOU LET THEM MAKE GDP
SOLUTIONS OR WILL YOU DO THAT
KNOWING THEY WILL SPILL SOME OF
IT AND YOU MIGHT BE LOSING A
POST DOC'S WORTH OF WORK OR YOU
GOING TO LET THEM MOVE TO TO THE
GLYCOSYLATION EXPERIMENT WHICH
IS THE IMPORTANT PART AND RERUN
THAT MULTIPLE TIMES,IME NOT SURE
THAT'S PART OF 1 OF YOUR LABS SO
IF YOU'RE HEART GOT PUMPING AND
YOU WILL SAY YES, THEN THERE'S
SPECIAL OPPORTUNITY HERE TO COME
IN AND DESIGN THE SUGARS AND LOW
TO BE AFARREDDED AND SOMETHING
THAT WILL KEEP FRUIT FLIES
HAVING A HEART ATTACK IF THAT
HAPPENS, SO OUR GRANT THEN WAS
TO MAKE THESE NUCLEOTIDE SUGAR
BLOCKS, SINCE THESE WILL BE USED
BY ENZYMES TO BUILD THESE, BUT
OTHER PRESENTER VS PUT UP THERE
ON THE SCREEN, AS A SPECIAL
ORGANIZATIONS NIEGZ EGG, WE ARE
INTERESTED IN SERVING
OPPORTUNITIES.
WE WANT TO MAKE THE PIs
COMFORTABLE THAT THEY WILL BE
ABLE TO GET A STABLE SUPPLY OF
THESE REAGENTS AND THEN ALSO BE
ABLE ABLE TO ENABLE THEM TO DID
THE EXPERIMENTS THEY WANT TO DO
WHERE THEY'RE NOT SPEND
THAGOREAN 70-75% OF THEST JUST
MAKING THOSE STARTING MATERIALS,
AND THEN ALSO AS A COMMERCIAL
ORGANIZATION, WE HAVE
ASPIRATIONAL GOALS, WE'RE
INTERESTED IN TRYING TO
FACILITATE KIND OF THE NEXT
GENERATION EXPERIMENTS WHERE
YOU'RE NOT WORRIED ABOUT THE
STARTING MATERIALS, YOU ARE
REALLY THINKING ABOUT THE ROOL
SCIENCE INSTEAD OF THE NUTS AND
BOLTS OF WHERE YOUR SUPPLIES ARE
COMING FROM.
SO THIS WAS A PARTNERSHIP
BUILDING OUT OF THE UO1 THAT WE
HEARD FROM FROM DR. LI, AND THEY
STARTED IN 2015 WITH THE CORE
SYNTHESIS, AND THE CORE OF HIS
TALK WAS ABOUT THAT CSY
EESYSTEM.
SO WHAT WE DID WAS A COUPLE
YEARS INTO THAT UO1 WAS WE
STARTED COMMUNICATING WITH THEIR
GROUP, THEY'RE GIVING US
INFORMATION ENZYMES WE'RE
PUTTING TOGETHER A COMMERCIAL
COLLECTION OF THOSE ENZYMES,
PROTOCOLS, BEING OPTIMIZED,
DEVELOPING PRODUCTS IN AN EVENT
DIVERSIFYING SOME OF THOSE
PROWCTDS AND AT THAT POINT WE
CAN FEEDBACK THEM BACK INTO THAT
ORIGINAL UO1 AND WE CAN HELP
THEM OUT, THEY CAN HELP US OUT.
PART OF THE REGION THEY
SEQUENCED THESE TALKS THIS WAY
IS IF YOU'RE OUT THERE, IF
YOU'RE THINK BEING THE TYPES OF
PROWCTDS YOU FLEED AND THOSE
GRANTS CAN YOU PARTNER UP WITH
THAT COMMERCIAL ORGANIZATION,
NOT ONLY FOR YOUR LAB BUT ALSO
FOR OTHER SCIENTISTS WANTING TO
WORK IN THE FIELD.
SO WE COMMERCIALIZE 11 COMMON
NUCLEOTIDE SUGARS.
THEY'RE COMMON TO MAMMALIAN
SYSTEMS, AND THESE WERE NOT
AVAILABLE IN 2017, SO WE'RE
ENDING UP WITH 7 UDP SUGARS, 2
GDP SUGARS AND 2 CMP, SO WHAT'S
COMMON ABOUT THESE REACTION
SYSTEM WE LIKE TO START WITH
ENZYMES FROM THE SALVAGE
PATHWAYS, WE'RE TRYING TO TAKE
SOME STARTING VERY CHEAP
SUBSTRATES, AND THEN FIRST
FOSTER NURSED FARALATE THEM WITH
A SUGAR KINASE AND THEN WE GO
AHEAD AND ACTIVATE THEM WITH A
SECOND ENZYME.
THESE REACTIONS ARE ALSO
INVOLVING AN ALCO LINE FOSTER
NURSED FOCUSED ON TAIS INHIBITOR
WHICH WILL PUSH THIS FORWARD,
TRY TO OPTIMIZE THE AMOUNT OF
PRODUCT WE CAN GET AND SPEED UP
REACTIONS, IF YOU HAVE SOMETHING
LIKE GDP GLUCOSE THAT YOU'RE
INTERESTED IN, IT HAS CHEAP
STARTING MATERIALS, SO CAN YOU
START HERE AS OTO THESE 1 POT
MULTIENZYME SYSTEMS.
SO I WANT TO GIVE YOU A CASE
STUDY, SO UDP, MY FAVORITE
NUCLEOTIDE SUGAR IS THE
ACTIVATED XYLOSE, WHERE IT'S
ATTACHED TO SOMETHING LIKE NOTCH
SIGNALING RECEPTORS, LARGE,
THINGS LIKE THAT, THIS IS ALSO
GOING TO BE THE INITIATION STEP
FOR CORE PROTEIN LINKAGE TO
MULTIPLE CLASSES OF PROTEIN
COMPLEXIO GLYCANS, THAT TEST HAS
TO GO ON BEFORE THE REST OF THE
TETRASACCHARIDE LINKER CAN GO
ON, IN CELLS IT WILL BE PRODUCED
IN THIS DE NOVO PATHWAY, THE
UDP, THEN ON THE SILOS, THIS NOT
THE WAY YOU WANT TO MAKE THIS IN
YOUR LAB.
THERE'S A REASON WHY THESE ARE
DID IN DIFFERENT COMPARTMENTS
INSIDE THE CELL, NADH WILL
INHIBIT UDH, IT WILL BE
REGULATED FOR THIS, SO THERE
NEEDING TO BE A BETTER WAY TO
MAKE THREEZ SUGARS.
FROM THAT PREVIOUS VERY BUSY
SLIDE, I TOLD YOU WE WANT TO
LOOK AT SALVAGE PATHWAYS SO
STARTING FROM FOZ FORALATION 1
POSITION, AND THEN EDITING IT IN
TIME.
SO IF WE WANT TO GET TO XYLOSRKS
E, WE WANT TO UTILIZE THIS
PATHWAY.
WE ARE ALSO INTERESTED IN THE
SYNTHESIS, WHERE WE CAN BI PASS
INTENDED THE FIRST STEP, AND THE
1, AND THEN SEE IF WE CAN
ENZYMATICALLY PUSH IT INTO THOSE
FINAL PRODUCTS.
SO WITH THAT, WE STARTED OFF
WITH 2 STEPS, TO GET US TO THE
SUGAR PHOSPHATE, WE HAVE A
[INDISCERNIBLE] HERE AND WE HAVE
THIS WITH THE RABBINOSE AND
XYLOSE, AND WE LOOK AT THE
CHEMICAL SYNTHESIS, WE END UP
WITH A ANAMERRIC MIXTURE, AND WE
HAVE EVIDENCE THAT HAPPENS AFTER
THE CHEMICAL PROCESSING BUT THEN
AFTER WE INTRODUCE THE ENZYME,
WE WILL SELECT FOR THE
BIOLOGICALLY RELEVANT ANAMERAND
WE ARE PRODUCING ENTIRELY
ALPHA-D XYLOSE IN THIS CASE.
BEING WE'RE A COMMERCIAL
ORGANIZATION, WE'RE INTERESTED
IN IN THIS MORE FEASIBLE AT
SCALE SO WE'VE BEEN ABLE TO
SCREEN ADDITIONAL ENZYMES AND IN
SOME CASES WOO CAN END UP WITH
MULTIPLE HITS, IN SOME CASES
JUST A SINGLE HIT BUT SOME OF US
TO OPTIMIZE THE INITIAL
ANALYTICAL PROOF OF CONCEPT,
THIS COMING OUT OF THE ACADEMIC
LABS.
BEYOND THAT WE'VE BEEN ABLE TO
TAKE THAT SAME TYPE OF STRATEGY
WHERE WE START OFF WITH THE
STARTING MATERIALS AND GO IN AND
THEN MODIFY THEM WITH ENZYMES SO
WE CAN CLEAN UP ANY OF THE
ANAMERRIC HAD OR OTHER MIXTURES
THAT WOULD HAVE COME FROM THOSE
SYNTHETIC PROCESSES, SO ALMOST A
CLEANING STEP AS WELL, SO ADD IN
KD--SALLY WHARKSAD IN CTP, THESE
ENZYMES AND THEN WE'RE ABLE TO
MAKE THE CMP ACID DERIVATIVES.
NOW THE BAM HALF OF THIS LIST IS
THINGS WE PRODUCED IN THE
HUNDRED MILLIGRAM SCALES, AND IN
YEAR 4 OF THE FAST TRACK AND IN
THE TOP HALF WE PREPARED THE
SUGARS AND IN THIS FINAL FIFTH
YEAR, WE'RE WORKING UP THE
SCALES OF THIS PRODUCTION ON
THIS AS L. SO AS WELL AS TRYING
TO BE COMMERCIALLY VIABLE, WE
ARE THINKING ABOUT THOSE
UNDERGRADUATES CAN WE PUT THINGS
IN KIDS, CAN WE GIVE THEM THE
OPPORTUNITY TO MAKE NUCLEOTIDE
SUGAR RIGHT THERE IN WHATEVER
REACTION THEY'RE DOING SO
EVERYTHING THEY NEED TO GET THAT
DONE, THEY CAN ADD THEIR OWN
TRANSFER ACE AND SEE IF IT'S
APPLIED TO THEIR PRODUCT OF
INTEREST, DERIFFITIZATION, SO IF
HAVE YOU YOUR OWN SUGAR, CAN YOU
POP THAT SUGAR IN THERE
ALONGSIDE WHAT WOULD THEN BE A
POSITIVE CONTROL WITH THE
STARTING SUGAR WE SUPPLIED IN
THE KIDS.
SAMPLER BACKS IF YOU GO TO
SIGMA, CAN YOU BUY A WHOLE
SEQUENCE OF NUCLEOTIDE TRI
POSFATES, WE WANT TO TRY TO ADD
NUCLEOTIDE SUGARS IN SAMPLER
PACKS.
AS WELL AS TRANSFER ACE, DO YOU
WANT TO TRY AN ENTIRE PANEL OF
TRANSFERAISES, TRYING TO BRING
DOWN THE COST BY PRINGING UP THE
PANEL THAT YOU WOULD HAVE ACCESS
TO.
SO I'M NOT ABLE TO COMPLETELY
DISCLOSE WHO WE WORK WITH BUT WE
DO HAVE ARRANGEMENTS WITH SOME
PEOPLE WHO ARE SUPPLYING
CHEMICALS AND OTHER RE18thS.
WE'RE ATLANTA SO WE CAN ALSO
WORK WITH BEVERAGE COMPANIES AND
WE'VE HEARD OF OTHER PEOPLE
TALKING ABOUT MMOs SINCE WE
HAD INTERACTIONS THERE AS WELL
AND SO THAT'S ONLY REALLY
FEASIBLE WHEN HAVE YOU THAT
LARGE STARTING MATERIAL, RIGHT?
YOU DON'T WANT TO BE ABLE TO SAY
DO YOU SOMETHING AND THEN YOU GO
TO THEIR WEBSITE AND YOU CAN'T
FIND THOSE NUCLEOTIDE SUGARS,
THEN YOU OF COURSE, THE LAST
SPEAKER WAS SAYING IF THERE ARE
THINGS THAT YOU ARE INTERESTED
IN, IF YOU ARE IN AN RO1, OR
PRODUCING THINGS THAT NEED
FEEDBACK ON YOUR PROJECT, CAN BE
USED BY OTHER PEOPLE WORKING IN
THAT AREA, THERE'S A GREAT
CHANCE FOR COLLABORATION THERE,
WRITING SBIR GROUPS AND WORKING
TOGETHER THAT WAY.
SO AFTER LUNCH, WE WILL HAVE
FREE SAMPLES OF HEALTHY TYPE
SUGAR, SOMETHING I WOULDN'T HAVE
SAID PRIOR TO 2017, YOU CAN
VISIT US AT CHEMILYGLIBBING O
SCIENCE AND MOVING AND THINKING
BO THE COMMUNITY, NOT JUST BEING
THE EXPERTS IN THIS ROOM BUT
ALSO OTHER BIOLOGISTS AND OTHER
GLYCO SCIENTISTS WHICH WILL SOON
BE REBRANDING AT CHEMILY BIO.
THANKY SO MUCH FOR YOUR
ATTENTION.
[ APPLAUSE ]
EMPLOY STHRKS I GOT A QUESTION
ABOUT THE AVAILABLE OF UDPIMRKS
ALNAC, HOPEFULLY THEY'RE
LISTENING IN TODAY.
SO OUR NEXT SPEAKER CONTINUES
THE THEME OF AUTOMATION.
DR. ALEXEI, DEMCHENKO,.
>> THANK YOU VERY MUCH DR. POHL,
IT'S MY PLEASURE TO BE HERE, I'M
SPEAKING TODAY ON NPI TEAM THAT
WAS IN CHARGE OF THIS PROJECT,
IS THERE AN ECHO SOMEWHERE?
CONSISTING OF MYSELF, KEITH
STIEN WHO WAS TYPICAL ATTENDEE
AT THESE MEETINGS AND CHRISTINA
[INDISCERNIBLE].
THIS WAS THE TITLE OF OUR GRANT.
MY LAB IS WORKING ON CHEMICAL
GLYCOSYLATION OLIGO SACCHARIDE
SYNTHESIS AND AUTOMATION, AND OF
COURSE CHEMICAL GLYCOSYLATION IS
A CHALLENGE WHEN WE WORK WITH
CHEMICAL GRID SERVICES
GLYCOSYLATION, WE NEED TO
CONSIDER EVERYTHING, LEAVING
GROUP, REACTING CELLS AND LIVING
GROUPS GIVE US TROUBLES AS
DR. WILS ORNAMENT YESTERDAY
NICEEE ILLUSTRATED WHAT
PROTECTION GROUPS CAN DO FOR US
AND SHE INTRODUCED 6 ORTHOGONAL
PROTECTING GROUPS.
'S ACTION ITSELF INVOLVES
PHYSICAL ORGANIC CHEMISTS ARE
ARGUING WHICH IS THE GOOD, BAD,
AND UGLY AND EVERYONE KNOWS THAT
UNCONTROLLED [INDISCERNIBLE] CAN
LEAD TO A MIX FUR OF ALPHABETTA,
AS WELL AS OTHER BYPRODUCTS THAT
BEEN PREDULTED FROM SIDE
PROCESS, IN MANY WAYS, TPHREU
RACING COSALATION, THE SINGLE
STEP GLYCOSYLATION WILL
CHALLENGE THOSE PEOPLE WORKING
WITH GLYCANS IN THE SPACE, AND
STILLFUL [INDISCERNIBLE] OF
WHOLE ASPECTS OF SYNTHESIS.
THERE ARE 2 GENERAL WAYS OF
MAKING TPHREU RACING KAN--KANAS
THESE DAYS.
MANUEL SIPGHT SIS THAT HAS MANY
ADVANTAGES IN TERMS OF STRATEGY
AND AVAILABLE, AND KNOW HOW
TECHNIQUES THAT WE,A PLIE AND
THERE ARE [INDISCERNIBLE] AS
WELL BECAUSE THEE CAN BE LENGTHY
AND ENZYMATIC SINCE PEOPLE CAN
PUBLISH 50 DIFFERENT OLIGIO
SACCHARIDES AND 1 ARTICLE
CHEMICAL SINCE PEOPLE PUBLISHED
1 ARTICLE PER SYNTHESIS.
SO AUTOMATED APPROACH IN THIS
RESPECT IS VERY ATTRACTIVE
BECAUSE COMPLETELY AUTOMATED,
APPROACH WOULD ALLOW YOU TO WITH
THE PRESS OF A BUTTON, DO
SYNTHESIS AND PRODUCE--PRODUCES
ACCURATELY, THERE ARE
CHALLENGES, THE CENSUS CAN BE
INCOMPLETE, IT'S HARDER TO
CONTROL IN GENERAL, HARDER TO
MONITOR.
LARGE BUILDING PLOK ACCESS MAY
BE NEEDED AND, WELL, ALL
EQUIPMENT AND EVERYTHING'S BEEN
DESIGNED FOR PEPTIDE SYNTHESIS
BUT NOT FOR GLYCAN SYNTHESIS AND
REGARDLESS THEIR APPROACH,
BUILDING BLOCKS AND LARGE PART
ARE MADE STILL MANUELLY,
ALTHOUGH FIRST AUTOMATION
EFFORTS HAVE EMERGED AND I
BELIEVE PROFESSOR PAUL WILL BE
SPEAKING RIGHT AFTER MY
PRESENTATION ABOUT AUTOMATION OF
BUILDING BLOCKS SYNTHESIS AND
FOR SURE WE WILL HEAR MORE FROM
CLAIR BENIT TOMORROW.
--OVER THERE AND PEOPLE IN THIS
AS WELL, TO STARTING TO DO
AUTOMATION AS WELL.
--THAT WOULD ALLOW FOR FULL
AUTOMATION.
THANKS TO NIH AND OTHER GRANT,
OUR LAB PURCHASED NUMBER OF
STATE-OF-THE-ART ADHAVE YOU VENT
SYSTEMS THATTA'S USED FOR
AUTOMATION THESE DAYS AND
GENERATION B THAT WOULD
DEVELOPED SHORTLY THEREAFTER,
INTRODUCED THE FOREIGN CHANGES
YOU SEE IN RED HERE, WE STARTED
USING LONGEST SPACER THAT WAS
PROVEN TO BE BETTER IN THE
INDEPENDENT STUDY, WE CHANGED
THE RESIN TO THE GEL WITHOUT A
PARTICULAR REASON AT THE TIME
AND ADJUVANT SYSTEM CAME WITH A
PUMP THAT GAVE US ADDITIONAL
CAPABILITIES AND AUTOSAMPLER WAS
USED FOR THE INSERTION TO THE
SYSTEM OF THE ACTIVATOR.
THE DONORS WERE CIRCULATED BUT
MANY ASPECTS REMAIN MANUEL
BECAUSE WE HAD TO STILL SWITCH
MANUELLY BETWEEN THE WASTE AND
DPLI KAN--KANA COLLECTION MODE
AND ALSO THE BUILDING--THE
BUILDING PLOKS WERE BOTTLES WERE
BUILDING BLOCKS WERE SWITCHED
WITH MANUEL 2, BUT THIS
GENERATION B WAS AROUND FOR A
WHILE AND WE USE IT FOR A NUMBER
OF SYNTHESIS, THIS DPLI
KAN--KANA STRUCTURE THAT WAS
ADDITIONAL CHALLENGE FOR US.
>> FINALLY WE FIGURE OUT TO YOU
DO IT WITHOUT AUTOMATION, WE
PUBLISHED PAPERS DESCROIBING OUR
EFFORTS IN IN RESPECT AND
COMPLETE AUTOMATION CAPABILITY
CAME WITH 2 WAY SLIT VALVE THAT
WOULD ALLOW US TO PROGRAM THE
MACHINE TO DIRECT THE FLOW
BETWEEN THE WASTE AND PRODUCT
COLLECTION MODE.
AND THE AUTOSAMPLER WAS REPLACED
WITH THE PREPRATIVE SAMPLER THAT
ALLOWED US TO INTRODUCE ALSO ALL
DOLLARS AND ALL REAGENTS
NECESSARY FOR ALL STEPS OF THE
SYNTHESIS.
THAT ALLOWED US TO REPURPOSE OUR
INTAKE PUMP LINES TO DELIVER
PURE SOLVENTS WHICH IS REALLY
IMPORTANT BECAUSE CAN YOU JUST
BY ACS GREAT, SOLVENTS AND USE
THEM AS THIS--33% YIELD WHICH IS
LOWER THAT BEFORE BY 12 HOURS IS
BECAUSE MY STUDENT WOULD IT IN A
WAY THAT IT DOESN'T NEED TO
TESTIMONY BACK IN OUR TIME, THIS
WAS USED PREVIOUSLY BECAUSE THIS
WAS SEEN, PRESS THE BUTTON, GO,
SLEEP, COME BACK IN THE MORNING
AND FINDED PRODUCT.
EMPLOY THE STUDENT DOESN'T HAVE
THE POSSIBILITY TO MONITOR THIS
REACTION BECAUSE WE DON'T HAVE
REMOTE ACCESS, IT'S EXPENSIVE
THESE DAYS, SO ANYWAY, THEY
PROGRAM THIS TO DO THE
GLYCOSYLATION.
IN ADDITION, AND OBVIOUSLY AT
THIS POINT WE LOST THE
CAPABILITY OF RECIRCULATING
WHICH IS A VERY IMPORTANT
FEATURE OF OUR TECHNOLOGY, MY
STUDENTS FELT OTHERWISE HE
REPLACED THE CIRCULATION OF
DONORS AND PRACTICES MOTIERS TO
USE WHEN NEEDED AND ALLOWED US
TO REPURPOSE THE SYSTEM WITH THE
USE OF ANALYTICAL AUTOSAMPLE
SINCE NOW THE ONLY SMALL AMOUNTS
OF DONOR AND PROMOTER WERE
INJECTED.
AND WE ALSO DEVELOPED OR OWN
RESIN THAT WAS NAMED AFTER THE
INVENTOR [INDISCERNIBLE]
GRADUATE STUDENT PICTURED HERE.
WAS BASED ON--FRIENDLY WINDOWS
BASED SOFTWARE AND WE CAN ALSO
MOVE TO TAKE A PEEK THROUGH THE
WINDOW WITH THE SAMPLES DOING,
IT'S ACCELERATED WINDOW AND IT
SHOWS THE DRAW SOME SOLVENTS
PREPARES MIXTURES AS WELL, AND
DRAWS AND PREPARES MIXTURES IN
THE SAMPLES SEAT, AND MIX AND
THEN INJECT AND REINJECT AND
BRING THE SYSTEM, IT NOW YOU SEE
THE DEVELOPMENT OF THE COLORS
AND THE ACID COMBINATION, NATIVE
AMERICANS IN THE NORTHERNINENCE
THERE DELIVERED TO THE SYSTEM,
AND THEN THE NEEDLE GETS CLEAN.
SO WHAT ELSE?
VERY RECENTLY WE SWITCHED TO
AUTOMATION IS SOLUTION, AND I
WILL TELL YOU WHY, FOR FIRST
ATTEMPT, WE REALLY DISCOURAGE
AND THE FIRST ARE THE
DIFFERENCES, PROCESS AGAIN, I
READ SINCE THE AUTOMATION AND
SOLUTION, AND IN THE COLUMN, WE
ARE STILL USING OUR
[INDISCERNIBLE] TO RECIRCULATE
WHERE WE DELIVER ACCEPTANCE FOR
SAMPLES.
SO THE FIRST ATTEMPT WAS VERY
DISCOURAGING--WITH THE DONOR IS
FAST AND 93% YIELD, SAMEON
DITIONS IN THE HBLC FAILED
MISERABLY AFTER 1 HOUR, WE HAVE
A 25% PRODUCT AND WE KNOW THE
DOCTOR'S ARTICLE THAT CAN YOU
FROM MANUEL TOA THE OTHER
APPROACHES MAY NOT BE SO
STRAIGHT FORWARD AND WE
STRUGGLED A LITTLE BIT UNTIL WE
WANT TO DO THE PLOTS ON THE
TABLE AND WE WILL LEARN HOW TO
MAKE IT AS EFFICIENT AS
[INDISCERNIBLE], ALSO ENGAGED
HIGH SCHOOL STUDENTS WHO
PRODUCED THE REACTION WITH THE
[INDISCERNIBLE] SO THE STUDENT
WHO WAS GRADUATING NOW SLEEZE IN
[INDISCERNIBLE]'S LAB, SHE DID
88% YIELD GLYCOSYLATION,
UNSTRAINED OFMORE HIGH COOL
REACTION AND PRODUCED WITH 84,
86 AND 86% YIELD.
SO IT'S NOT REALLY AN ONERATED
DEPENDENT PROCEDURE ONCE IT'S
PROBABLY REFINED.
SO NOW WHY ARE WE SO INTERESTED
IN SOLUTIONS SUDDENLY, OF COURSE
NOT GIVING UP ON THE POLYMER
SUPPORTED SYNTHESIS BUT SOLUTION
BASED APPROACHES WERE DEVELOPED
DURING THE PANDEMIC BECAUSE THE
WAY WE DO RESEARCH CHANGED
SOMEWHAT, OF COURSE WE'RE
TOTALLY UNPREPARED, WE PANICKED
BUT EVEN WHEN WE CAME BACK TO
LABS WE STARTED OPERATING WITH
REDUCED HOURS, TIME SHARE ALSO,
IT WAS IMPORTANT FOR US TO LOOK
AT BATCH SYNTHESIS WITH A PRESS
OF A BUTTON WE CAN MULTIPLE
POLARIZED KIEWLS, SET THE
MACHINE, LEAVE, LET OTHERS IN
AND THE STUDENT WOULD COME BACK,
THEY WILL GET MULTIPLE COMPOUNDS
SYNTHESIZED IN THE ABSENCE.
SO IN THIS EXAMPLE, OVER HERE WE
HAVE ONLY 1 DONOR FOR ACCEPTORS
THAT COULD ALL BE INGESTED AND
IN A MATTER OF I THINK IN THIS
CASE, ABOUT 8 HOURS, THE STUDENT
WILL COME BACK AND FIND FRACTION
COLLECTION ALL THE PRODUCTS
SYNTHESIZED, YIELDS ARE NOT
STELLAR BUT DON'T FORGET THAT'S
WHERE WE'RE TALKING ABOUT
COMPLETE AUTOMATION HERE AND TO
BE HONEST SOME OF THESE YIELDS
ARE OKAY IN MOST PARTS.
OTHER EXAMPLE, WHEN THE STUDENT
RANDOMLY PROGRAMMED A
COMBINATIONS OF DONORS AND
ACCEPTORS, THEY ARE ALL LOADED A
FEW OF EACH AND BY RANDOMLY
PROGRAMMING COMBINATIONS, THE
NEXT MORNING AFTER 12 HOUR
SEQUENCE MY OUTPATIENT SETTINGS
[INDISCERNIBLE] SHE FOUND 6
DIFFERENT DISCIPLINARY
SACCHARIDES AND TRI SACCHARIDES
IN THE COLLECTORS.
AS DR. POHL MENTIONED, WE ALSO
SYNTHESIZED STRUCTURES IN THE
SOLUTION BASEDDA, PROACH THAT
ARE NOT PICTURED HERE, WE DID
THIS AS WELL, THIS WAS NOT
EXTREMELY EFFICIENT AND THIS IS
1 OF THE FOCUSES IN OUR LAB
THESE DAYS, CAN CHRISTINA DEMILL
WE WORKING ON DECORATING THIS
AND IT'S ALL DONE NIEWN
CHEMICALLY.
THIS PROJECT RESULT INDEED 28
PUBLICATIONS AND THE MOST RECENT
1 WAS THANKS TO JOE BARKY WHO
INVITEDITOUS SCRIEWBT TO THE
NICE COLLECTION OF GLYCO SCIENCE
ARTICLES AND THIS DESCRIBES OUR
GENERATION A, B, & C,
GENERATION D IS IN THE WORKS AND
I LOOK FOR MORE DEVELOPMENTS IN
THIS DEAL.
SO WHAT CAN YOU DO WITH OUR
TECHNOLOGY, WE DON'T SELL
ANYTHING BUT IT'S ALL FREELYY
AVAILABLE.
SO YOU ARE WELCOME TO READ OUR
PAPERS AND [INDISCERNIBLE].
TO FINISH THEN, SINCE MICROPHONE
FELL.
[LAUGHTER]
WE COULDN'T HOAFORT MANY
STUDENTS THESE DAYS BUT WE HAVE
1 FROM ITALY WHO WILL BRING
THEIR TECHNOLOGY TO THE OLD
WORLD SO ANYWAY, IF YOU HAVE
HPLC AND MANY OF YOU DO WAYED ON
YOUR PRESENTATIONS CAN YOU DO
THE SYNTHESIS, CAN YOU REPURPOSE
SYNTHESIS TO SEPARATE IF WANT TO
ORE CAN YOU BYPASS THIS.
ALL RIGHT, CAN THAT, THAT'S ALL
I HAVE TO DO, IS TO THANK COMMON
GROUND, TEAM, THIS IS A PHOTY, A
DIFFERENT PHOTO EVERYONE ELSE IS
SHOWING SO I PULLED IT FROM MY
FIRST MEETING WHICH WAS A SECOND
YEAR OF COMMON FUNDS BECAUSE MY
COLLABORATORS AND MY GROUP,
THANK YOU SO MUCH.
[ APPLAUSE ]
>> WHERE IS AUTOMATION MOST
VALUABLE?
IS IT IMET TORE HAVE SINGLE STEP
AUTOMATIONS LIKE HE JUST SHOWED
NEAR THE END OF HIS TALK WHERE
ALL THE TPHREU RACING
COSALATIONS ARE AUTOMATED
BECAUSE THEN HAVE YOU THE ACCESS
TO THE DISCIPLINARY SACCHARIDE
OR PENTAWHATEVER IT MIGHT BE SO
CAN YOU PUT IT INTO THE NEXT
LIBRARY STEP.
IT SEEMS LIKE ALEX SESTARTING TO
GO TOWARD THE SECOND 1 WHERE
THERE'S A LOT OF VALUE IN
INDIVIDUALLY AUTOMATING EACH
STEP AND I HAVE TO SAY THAT'S
WHERE MY OWN LAB IS ALSO
STARTING TO WEIGH IN BECAUSE IN
TERMS OF BROAD UTILITY, THERE'S
SO MUCH DIVERSITY IN GLYCANS, BY
ONLY MAKING A SINGLE
PENTA-WHATEVER SACCHARISE AS
OPPOSE TO AUTOMATING EACH OF THE
INDIVIDUAL STEPS WE LOSE
FLEXIBILITY OVER ALL AND THAT'S
1 OF THE REASONS WHY WE'RE
STARTING TO LOOK ALSO AT HOW DO
WE AUTOMATE INDIVIDUAL STEPS.
SO 1 OF THE THINGS YOU MAY NOT
APPRECIATE FOR THOSE OF WHO YOU
HAVE BEEN THINK THISSING ABOUT
AUTOMATION FOR DECADES MY DAD
WAS A TEXTILE WORKER THERE WAS 1
OF THE FIRST INDUSTRIES THAT WAS
AUTOMATED HUNDREDS OF YEARS AGO
SO I ALWAYS GREW UPON WITH THE
IDEA OKAY, YOU NEED TO DO
SOMETHINGALUE ADDED AS A HUMAN
BEING, THE MACHINES CAN DO THE
REGULAR LABOR.
WHAT'S AMAZING IS HOW CHEAP THEY
BECOME.
YOU WILL SEE HPLC PUMPS OVER AND
OVER AGAIN, USELESS THE ABILITY
TO CONTROL THOSE WITH MUCH
SIMPLER SOFTWARE THAN WHAT WAS
AVAILABLE 20 YEARS AGO, WHEN I
STARTED IN 2000, IN MY CAREER
AND WANTED TO HOME BUILD A
SYNTHESIZER, I WENT BE OVER TO
THE ENGINEERING SCHOOL AND
THEY'RE LIKE THIS IS WAY TOO
COMPLICATED, THIS IS TOO
COMPLICATED AND NOW WHAT WE'RE
SEEING IS THAT THE AUTOMATION
THAT'S ALREADY AVAILABLE, WE CAN
MIX AND MATCH THIS, WITH THE
HPLC PUMPS, PYTHON CODING WHICH
DIDN'T EXIST 22 YEARS AGO, MAKE
ITS ALSO EASIER TO HOME BUILD
THINGS WITHOUT HAVING A LOT OF
CODING BACKGROUND.
SO WITH THAT, I WANT TO GIVE YOU
A LITTLE BIT OF SENSE FOR WHAT
WE'VE BEEN DOG IN LOOKING AT
THIS.
AS I SAID YFULT BUT THE IDEA IS
TO BE ABLE TO PUSH FORWARD THE
METHODS FOR BOTH [INDISCERNIBLE]
ANALITICAL CHEMISTRY AS WELL AS
ANALYTICAL 60 SIS, THESE ARE ARE
ALL VERY MUCH INTERLINKED, THESE
ARE STANDARDS WE MADE THROUGH
SYNTHESIS, AND WE ISOLATE OR
NATURAL RESOURCES THROUGH
ENZYMATIC RESOURCES, YOU DRIVE
THE ANALYTICAL STANDARDS, CAN
YOU ALSO RULE OUT STRUCTURES,
ESPECIALLY ISOMERS BASED ON
DIFFERENT PROPERTIES THAT ARE
HARD TO PREDICT DE NOVO BUT IF
HAVE YOU AUTHENTIC SEASMS YOU
CAN DEVELOP CHROMATOGRAPHY
PROTOCOLS OR MASS SPEC THAT CAN
DIFREBTIATE SPECTERS THAT'S A
FRONTIER THAT WE'RE OPENING UP
AS CHEMISTS BECAUSE WE CAN NOW
MORE EASILY GET TO THE ISOMERS,
THE ALPHA, BETA, DIFFERENT
ISOMERS ALL OF WHICH HAVE EXACT
SAME MOLECULAR WEIGHT IN THE
MASS SPEC BUT NOW CAN YOU
DIFFERENTIATE THOSE BY THE
ISOMERIC RATIOS.
SO THIS LOOKS LIKE THE WRONG
TALK.
THIS LOOKS LIKE YESTERDAY'S
TALK.
SO WHAT DO YOU NEED, SO WE'VE
SEEN MULTILE DIFFERENT
AUTOMAITIONZ PLATFORMS NOW AND
WHAT BRINGS ALL THESE TOGETHER
IS THE ABILITY TO DECIPHER A
LIQUID HANDLING, A LIQUID
HANDLING SYSTEM, YOU CAN DELIVER
SLURYS AND IT CERTAINLY IS DONE
INDUSTRIAL BUT IT IS CHALLENGING
THROUGH THE SIZE OF THE SLURY
ARTICLES CAN ANOTHER VERYIABLE
YOU HAVE TO CONTROL, SO REALLY
THE IDEAL PART IS TO BE ABLE TO
AND TO PUT EVERYTHING INTO
SOLUTION, YOU HAVE MULTIPLE WAYS
OF HAVING AUTOMATED DELIVERY
WHETHER THAT'S THROUGH SERIES
POINTING PUMPS FOR SMALLER SCALE
OR THROUGH THE HPLC PUMPS THAT
WAS JUST SHOWN IN THE PREVIOUS
PRESENTATION, YOU THERE ARE MANY
WAYS CAN YOU GET TO THE LIQUID
HANDLING BUT THEN YOU THINK
ABOUT THE CHEMISTRY ITSELF, IF
YOU WANT TO DO SOMETHING WITH
THE PUMP, HOW DO I DO THAT WITH
THE CHEMIST RADIOY IN THE
LITERATURE FOR THE LAST 150
YEARS OR SO BECAUSE CHEMISTS
HAVE USED THIS IDEA OF DRIVING A
REACTION FORWARD, OFTEN TIMES BY
A PHASE CHANGE WHICH IS EXACTLY
WHAT DO YOU NOT WANT IN AN
AUTOMATION PLATFORM, SO A LOT OF
THE CHALLENGES AS WAS JUST
ALLUDED TO BEFORE, IS THAT WHEN
WE DO SOMETHING MANUELLY WE
DON'T CONTROL A LOT OF HEAT AND
MASS TRANSFER ISSUES AND WE
OFTEN TIME VS PURPOSELY DESIGNED
THE REACTION TO DO A FACE
TRANSFER CHANGE, THAT PLEENS
THAT WE CAN'T AUTOMATICALLY JUST
TRANSFER IT INTO A LIQUID
HANDLING SYSTEM.
SO WE STARTED OKAY, THERE WERE
SOLUTION IN SOLID PHASE METHODS
OUT THERE TO DO DISP WHAT DO WE
NEED TO AUTOMATE.
AS LONG AS YOU HAVE YOUR
SOLUTION PHASE REACTIONS, YOU
CAN PROGRAM YOUR 2 BUILDING
BLOCKS TO COME TOGETHER JUST
LIKE YOU DO WITH NUCLEIC ACID OR
PEPTIDEY 60 SIS BUT BRUCE WHO
INVENTED THIS IDEA BACK IN THE
1950S AND 60S FOR PEPTIDE
SYNTHESIS ALSO HAD THE PROBLEM
OF PEPTIDES WHEN YOU COUPLE THEM
ONCE THEY GET LONGER IS START
SELF-AGGREGATING AND
PRECIPITATING OUT OF SOLUTION,
SO THIS PROBLEM WAS HE COULD BT
MAINTAIN SOLUBLE OF HIS PEPTIDES
AS THEY GROW BUT AS MANY EVER
YOU KNOW ONCE YOU PROTECT A
SUGAR YOU DON'T HAVE THE SAME
PORT OF PROBLEMS IN BASICALLY
HAVING YOUR SOLUTIONS, YOUR
SUGARS THEMSELVES PRECIPITATING
OUT OF SOLUTIONS, IT'S USUALLY
REAGENTS THAT PRECIPITATE OUT OF
VARIOUS WAYS.
SO THOUGHT WE DON'T NEED TO USE
THE OLD 19 NIFULTS OF WE COULD
USE SOLUBLE PHASE AND IF WE
COULD USE TAGS AND WE LOOKED IN
THE LITERATURE, WE SAW LIPID
TAGS AND COULD WE DEVELOP A
SYSTEM THERE, IF YOU WANT TO DO
A SINGLE REACTION, AS THEY
SHOWED TOWARD THE END, YOU DON'T
NEED TAGS AT ALL AND IN FACT
WE'RE START TO THINK IN WITH
THE'M PROVEMENTS IN
CHROMATOGRAPHY THE LAST 20 YEARS
WE MIGHT NEED TO RETHINK OUR
FLUORIST TAGS, DO WE NEED THEM
AS WE SAW WITH OUR
COLLABORATION, THERE'S A LOT OF
VALUE IN STILL USING THAT TAG
FOR THINGS LIKE SULFATED
COMPOUNDS WHERE IT'S EASIER TO
FESH OUT.
SULFATE COMPINEDS ARE DIFFICULT
TO PURIFY USING STANDARD HPLC
METHODS.
THE PROBLEM WITH THESE TAGS OUT
THERE.
SO A RIP EDUCATIONAL TAG, YOU
WOULD SEE, AND THEN YOU TRY TO
PICK OUT THINGS IN THE NOISE
PRACTICALLY WITH YOUR SUGAR, SO
WE SAID LIPID TAGS AREN'T THAT
USEFUL IF WE WANT TO MONITOR
REACTIONS AS WE GO ALONG, SO
WHAT ABOUT POLYETHYLENE TAGS
THEY HAD BEEN USED WITH THE
CHEMISTRY DEVELOP THE IN THE
1990S AND THEY'RE BASICALLY 2
CARBONS LINK WIDE OXYGEN.
AND THOSE OF YOU ARE USED TO
LOOKING AT NMRs, KNOW THAT
THAT'S EXACTLY LIKE AN OXYGEN
LINK TO A CARBON AND SUGAR AND
INDEED YOU LOOK AT THE NMR AND
THE TAG OVERLAPS EXACTLY WITH
THE SUGAR.
SO YOU THOUGHT, WE NEED
SOMETHING DIFFERENT AND
[INDISCERNIBLE] HAD BEEN
STARTING TO USE USE LITTLE
PIECES OF TEFLON, TAGS THAT THEY
GET ATTACHED TO ORGANIC
COMPOUNDS TO MAKE LIBRARIES AND
WE THOUGHT, YOU KNOW IN THE
SUGAR CASE, THIS MIGHT BE EASIER
BECAUSE WOO VERMEN INFECTED A
LOT OF OXYGENS ON OUR SUGAR THAT
ALLOW THIS TO DESIRED
[INDISCERNIBLE].
AND THAT WOULD DISCOVER THAT WE
COULD HAVE LARGE GROWING
CARBOHYDRATE CHAINS AS WELL AS
HAVE THE FLUOREST TAG AND STILL
BE ABLE TO PURIFY OUT OUR
COMPOUND.
NOW IF WE WANT TO AUTOMATE THIS,
OF COURSE THIS HAD TO BE
PROCESSED WHERE WE HAVE TO
REINVENT THE YOUR PARTICULAR
COMPOUND.
YOU CAN'T AUTOMATE A PROCESS OF
EVERY SINGLE SUGAR CAN YOU TO DO
A DIFFERENT PURIFICATION
PROTOCOL.
THE OTHER THING WE HAD TO SEE
WAS WHETHER ASHES TACHING A
LITTLE C817 TAGGED TO OUR SUGAR
WOULD ALLOW US TO ACTUALLY BE
ABLE TO DO THIS KIND OF
CHEMISTRY AND DO A PURIFICATION
SEPARATION REGARDLESS OF WHAT
THE TAG MIGHT NEED.
AND WE FOUND OUT THAT WE CAN
RERNTION GARDLESS OF THE SUGAROT
FLUORIST TAG AND NOT OHM THAT
ONCE WE LOOK ATOTMATION
PLATFORMS, LOOKING IN AT CHEM
SPEED THAT WE MACHINE SHOP TO
MODIFY, IT TURNS OUT BECAUSE
THAT FLOOR IS GEL THAT THE FIRST
TACK ALLOWS TO YOU DO THE
SEPARATION DOESN'T STICK TO
OTHER COMPOUNDS THE WAY OUR
NORMAL 1 DOES, AND REACH THAT
SAME OVER AND OVER AGAIN, SO 1
THING MANY DON'T APPRECIATE IS
THE REASON WHY WE COULD AUTOMATE
THIS PROCESS IS BECAUSE THE THIS
RINSES SO CLEAN THAT WE COULD
GET AWAY WITH THE PROCESS.
SO YOU SEE THE MACHINE WITH
THIS, WE HAVE THE BUILDING PLOKS
THAT WE NEED TO BE ABLE TO GET
TO THOSE, WE NEED REAGENTS TO
COUPLE THE SUGARS THEMSELVES AND
WE NEED A PURIFICATION SYSTEM
FOR THIS SORT OF MULTISTEP
SYNTHESIS.
SO THIS IS WHAT WE ULTIMATELY
DEVELOPED THAT IT INHIBITED
THEIR ABILITY TO MOVE ON TO MAKE
THE DPLI KAN--KANAS THEMSELVES.
THIS IS A BIT OF A PICTURE TO
SHORTENED WHAT WE'RE SEEING HERE
BUT THE ROUGH UPPER LEFT HAND
CORNER YOU SEE THE NEEDLE ARM
AND HAD IS THE BASIC PROCESS FOR
ALL THESE DEFINITE AUTOMATION
PROCESSES THIS IS A NEEDLE ARM
THAT TAKE THE SOLUTION OUT OF A
CONTAINER JUST LIKE THE HPLC IF
YOU HAVE A RESIDENT IN A
CARTRIDGE, SO WE CAN GO DOWN TO
MINUS 40-DEGREE FIST WE WANT TO
DO LOW TEMPERATURE REACTIONS,
AND WE CAN REFLEX WE WANT TO,
ALTHOUGH IT'S LESS USEFUL, SO UP
THERE IS THE EXTRACTION MEDIAN
FROM IT.
SO THIS IS THE ACTUAL PROCESS,
WE TAKE THE COMPOUND AND WE PUT
THEM TOGETHER AND LOAD IT ON TO
OUR SILICA GEL AND THEN BY
TAKING OUR AQUEOUS MIX THUR
ALLOWS THE INTERACTION FOR THE
COP BINNATION TO ADHERE TO THE
COLUMN AND EVERYTHING ALOWTS OUT
AND THEN WE USE A PURE ORGANIC
SOLVE TONIGHT ALLUDE OFF OUR
DESIRED COMPOUND AND SO OF
COURSE WE ARE DOING THIS FIRST
WITH NEUTRAL COMPOUNDS.
AND STARTED LOOKING AT THIS IN
VARIOUS THINGS.
HERE'S AN EXAMPLE OF WHAT I WAS
TALKING ABOUT BEFORE.
THE FLOR --FLOR, TAG AND IS
INCORPORATED.
CAN WE INCORPORATE A SULFATE
INTO THE PROTOCOLS AND WOULD WE
BE ABLE TO DO THE SOLID PHASE
EXTRACTION OR WOULD NOW, THAT
TEGGIC--STRATEGIC 5LY CHARGED
SULF 8 GO THROUGH IN THE WATER,
IT WOULDN'T ADHERE TO THE
EXTRACTION COLUMN.
AND VICTORIA AN NIH POST DOC
FELLOW AT THE UNIVERSITY OF
UTAH, SHE WAS LOOKING FAVORITE
CAN WE GET TRANSFER PROCESS AND
IT URN ITS OUT THE COMMON
SULFATION PROTOCOLS THAT WE USE
AT THE KONSZITRATIONS
THAT--CONCENTRATIONS WHEN YOU
CAN CAN'TAD AID SOLID INTO YOUR
REACTION, AND YOU SART OUT WITH
A SALVATIONARMYUSAENT AND ADD
SOLUTION AND IT DESOLVES.
AFTER TRYING MANY THINGS I
ENCOURAGE HER TO LOOK AT THE
PAPER PER DETAILS, AND SHE
DISCOVERED THAT SHE COULD USE TO
BE ABLE TO DEVELOP THIS
AUTOMATION PROTOCOL AND IN THIS
CASE, MAKE THESE SOLVATED RAMINS
AND YOU COULD HAVE THE SOLVATED
COMPOUND STICK TO THE PHASE,
EXTRACTION MEDIA AND DO THIS
KIND OF PURIFICATION PROTOCOL
AND THAT WAS THE STARTING POINT
FOR THE FANTASTIC COLLABORATION
WITH LINDA WILSON'S LAB THAT YOU
HEARD ABOUT YESTERDAY IN BEING
ABLE TO APPLY THIS SORT OF
CHEMISTRY TO THE GLYCO IMMUNO
GLYCANS.
WE ARE ALSO LOOKING AT DEFINITE
PROTOCOLS AND I WON'T INTO ANY
DETAILS BUT WE ALSO HAVE BEEN
WORKING ON INCORPORATING A WIDE
VARIETY OF CHEMIST RADIOYS INTO
THIS PROTOCOL AND WE HAVEN'T
SEEN A LIMIT TO THE SOLID PHASE
EXTRACTION TAG IN TERMS OF
CHEMISTRIES THAT WE'RE DOING, IT
IS ELECTRON WITH DRAWING SO WE
HAVE A BIT OF A SPACER BETWEEN
THE CF817 AND OUR SUGAR BECAUSE
OTHERWISE IF IT'S TOO CLOSE IT
WILL AFFECT THE CHEM RIEOF THEY
AS CAN YOU IMAGINE, BUT
OTHERWISE THIS COULD WORK QUITE
NICELY.
WE ALSO AS I MENTIONED YESTERDAY
HAVE BEEN WORKING ON TRYING TO
PUT GLYCO [INDISCERNIBLE].
OTHER DIFFERENT TYPES OF CHEMIST
RADIOYY THAT YOU DON'T GET IN
NATURAL PROWCTD PLODUCTS AND
WOULD BE IMPOSSIBLE TO DO WITH
THE CHEMISTRY.
LIKE I SAID YESTERDAY, WE ARE
WORKING TOGETHER SO QUEE FOLK
THE PROBLEM WE CAN DO WITH THE
NATURAL ISOLATION OR ENZYME
CHEMISTRY AND CERTAINLY MAKING
THIS KIND OF SULFUR LINKAGE FOR
STABILITY AGAINST ENZYMES
CLEAVAGE IS 1 OF THOSE KINDS OF
CATEGORIES AND ONCE AGAIN IT
DEPEND OFFICE OF DIVERSITY BEING
ABLE TO FIND SLIGHTLY DIFFERENT
CHEMIST RADIORYS AND PROTOCOLS
TO GET THIS LINKAGE TO COME IN.
WE COULD APPLY IT TO OTHER
DIFFERENT KINDS OF SUGARS.
SO I SAID YESTERDAY, THIS IS ALL
GREAT BUT THE REASON WHY WE ARE
STILL MAKING HUGE AMOUNTS IS THE
BUILDING BLOCKS STATION AND WHY
CAN'T WE AUTOMATE THESE?
WHY IS IT THAT WE'RE DOING ALL
THESE STEPS MANUELLY THEN WE
MAKE COMPOUNDS AND THAT'S WHAT
MAKES THE AUTOMATED BUILDING
CLOCKS OF DEC NOT JUST THE DPLI
KAN--KANAS THAT THESE THEMSELVES
BECAUSE OTHERWISE WE DON'T HAVE
AUTOMATION DPLI KAN--KANA
SYNTHESIS.
AND SO, THIS IS WHERE WE
TRANSATION, SOME OF YOU ASKED ME
THIS UNIVERSITY, SO I THOUGHT IT
WAS WORTH A SLIDE HERE, WE'RE
USELY DOING THINGS, FOR
ENSIKEATIC CHEMIST RADIOY, THAT
MEANS CAN YOU STILL USE LIQUID
HANDLING TO PUT BOTH YOUR
COMPOUNDS INTO THE GLASS REACTOR
BUT THEN YOU'RE LIMITED TO BY
THE SYSTEM OF THE REACTOR, WE
COULD DO A PARALLEL REACTION BUT
THERE'S EMILY THE TO THE SCALE
BASED ON THE REACTOR.
BUT IF YOU LOOK AT THE CHEMICAL
INDUSTRY ESPECIALLY PROCESSING
NATURAL GAZ AND THE GASOLINE,
OIL PRODUCTS THEY DO THAT IN
FLOW.
THAT THAT YOU BASICALLY TAKE
YOUR STARTING MATERIALS AND THE
REACTION HAPPENS IN THE TUBE AND
IT STATES CONSTANT BECAUSE THE
REACTION IN THE SAME
[INDISCERNIBLE] ULTIMATELY.
NOW YOU HAVE TO RETHINK
CHEMISTRY BECAUSE NOW YOU WANT
FAST, YOU DON'T WANT TO HAVE TO
CHUG THIS ALONG SO SLOWLY IN A
TUBE THAT YOU'RE RUNNING
REACTIONS FOR 5, 6, 7 HOURS AND
FLANKLY ON THE AUTOMATION
PLATFORM, WE HAVE THE REACTION
THAT MORPHS FOR POWERS BECAUSE
THE FLAT FORM IS MONEY AND SO
LESS TYPE HAVE YOU ON THAT, THE
BETTER THE PROCESS BECOMES
OVERALL AND SO, SCALING REACTION
TO MATCH, HAS A LOT OF PATHWAY
GIVES RAMSTERS AS WE ORDER FROM
THE PREVIOUS GROUP, IT'S
CONTINUOUS FLOW, ONCE HAVE YOU
THE PROCESS YOU CAN RUN IT FOR A
PERIOD OF TIME AND THEREBY GET
MORE PRODUCT, SO YOU DON'T HAVE
TO DO REOPTIMIZATION, AND THIS
IS OUR PATHWAY TO THE 15 YEAR
GOLD TO VERY QUICKLY AND EASILY
GROW AN INDUSTRY AROUND THIS
WHICH IS WHAT I WAS EXCITED
ABOUT HIDO WE GROW THESE AROUND
THIS TO BE ABLE TO SCALE
EVERYTHING THAT WE MAKE AND THIS
IS THE ANAGRAM OF FLOW CAM IS
THE OPEN SOURCE SOFTWARE TO
PROGRAM THESE THESE.
SO WE TALKED ABOUT THIS ALSO
AFTER WE WORK WIDE CLAY BENIT
AND YOU WILL HEAR MORE ABOUT HIS
WORK LATER.
WE NOW HAVE THESE PROCESSES,
WHERE WE CAN COMBINE PUMPS, THE
SYRINGE PUMPS OR HPLC PUMPS, WE
CAN DO ORTH1 REACTION OR WE CAN
FIEBD COMMON SELF-ENTS SO WE CAN
[INDISCERNIBLE] REACTIONS AND
START MAKING ENTIRE BUILDING
PLOKS FROM FLOW AND THIS, YOU
KNOW IT TOOK THEM ABOUT A WEEK
TO MAKE THIS MANUELLY AND
BASICALLY A MORNING TO MAKE THIS
IN A FLOW PROCESS, OBVIOUSLY YOU
HAVE TO RUN IT LONGER IF YOU
WANT TO DO IT ON A KILOSCALE BUT
THE IDEA IS THAT YOU SAVE A LOT
OF LABOR AND HALF A DAY TO MAKE
A BUILDING PLOOK AND WHAT I WANT
BECAUSE IF I HAVE AN IDEA OR
HAVE YOU AN IDEA, OR WEEKS TO
MAKE THE BLOCK BEFORE WE GET TO
THE GLYCAN SYNTHESIS.
SO THIS IS THE FIRST EXAMPLE OF
AUTOMATED CARBOHYDRATE BUILDING
PLOKS SYNTHESIS AND WE'RE
EXCITED TO KEEP GOING WITH THIS
PROCESS TO BE ABLE TO AUTOMATE
ALL SORTS OF DIFFERENT BUILDING
PLOKS, INCLUDING THE 1S INVOLVED
IN PEPTIDE O DPLI KAN--KANA
SYNTHESIS AND THAT'S WHAT WE
HEAR ABOUT NEXT IS HOW DO WE USE
SOME OF THESE PROWCTDS THAT GEP
ANALYTICAL TECH NEEBS BECAUSE WE
HAVE AUTHENTIC SAMPLES AND
FINALLY I WANT TO MAKE SURE IT'S
VERY CLEAR, AUTOMATION REQUIRES
HUMAN CREATIVITY, THE ICS SIGHT
8 HOURS THING ABOUT AUTOMATION
IS YOU CAN GET RID OF A LOT OF
TEDIOUS WORK, CLONING IS CHEAPER
NOW THAN BACK IN THE 1700S WHEN
EVERYTHING HAD BE MADE MANUELLY.
THAT'S THE SAME PROCESS I SEE
HERE, WE CAN SHIFT OUR LABOR TO
TEDEIUM LABOR TO THINK ABOUT
PROBLEMS AND DEVELOPING
CHEMISTRY AND QUICKLY DEVELOP
AND TRANSFERRING THAT TO OTHER
LABS.
SO WITH THAT, I THANK YOU FOR MY
FORGZ OF THIS PORTION AND THEN I
WOULD LIKE TO TRANSITION TO
DR. KRISTI PETERSON'S WORK WHO
WE'VE BEEN WORKING WITH AS PART
OF A SUPPLEMENT TO LOOK AT
PEPTIDE DPLI KAN--KANAS AND
WHERE WE'RE WORKING ON BUILDING
DPLOK SYNTHESIS PERLET PEPTIDE
GLYCAN PORTIONS, THE BUILDING
BLOCKS.
THANK YOU.
[ APPLAUSE ]
--SO THIS IS A PROJECT
SUPPLEMENT AS YOU HEARD FROM
NIKI AND I WORKING WITH 2 OTHER
MICROBIOLOGISTS [INDISCERNIBLE]
JUTRAS AND MARIA GOOD WHAT.
SO THE RO1 WAS BASED ON A
PROJECT TO BE ABLE TO DEVELOP A
RESERVOIR TARGETED VACCINE SO I
WILL STEP BACK FOR A BRIEF
SECOND AND SAY, THIS PROJECT IS
REALLY THE PERFECT POSTER CHILD
OF WHAT THE SUPPLEMENTS WERE
CREATED TO DO SO I AM A
VETERINARIAN WHO STUDIES DISEASE
IN POPULATION SAYS AND WHEN SHE
SAYS INVIVO SHE MEANS IN CELLS
AND WHEN I SAY INVIVO, I AM
TALKING ABOUT A CREATURE, SO I
AM LITERALLY THE FAR END OF THE
SPECTRUM LOOKING AT POPULATIONS
OF HUMANS, DOGS, OTHERWISE, AND
OF COURSE AS YOU'VE BEEN HEARING
FOR THE LAST DAYS YOU'RE LOOKING
AT GROUPS OF MOLECULES, SO IT'S
WONDERFUL TO BE ABLE TO BRIDGE
THIS TECHNOLOGY OVER TO MY
GROUP.
SO A BIT OF AN EXPLANATION OF
WHAT WE'RE DOING, MA RIA HAD
CREATED AN OUTER SURFACE PROTEIN
[INDISCERNIBLE] THE CAUSATIVE
AGENT OF LYME DISEASE, VACCINE
THAT WHEN MICE EAT AND WHEN MICE
EAT IT AS SHOWN HERE THEY THEN
CREATE ANTIBODIES AGAINST THAT
PROTEIN WHICH WHICH IS THEN
TAKEN UP BY THE TICK THAT
CARRIES LYME DISEASE.
THAT TICK THEN LOSES THE CANNED
WHAT, THEY ARE KILLED AND BY
CLEARING IT IDENTITY OF THE
TICKS, WE PROPOSE IN ON OUR RO1
TO SEE IF WE CAN THEN PREVENT
INFECTION OF BOTH HUMANS BUT AS
A PROXY WE ARE USING A GROUP OF
HUNTING DOGS.
IN ORDER TO DO THIS BETTER WE
REALLY WOULD LIKE TO HAVE BETTER
DIAGNOSTICS SO THE CURRENT
DIAGNOSTICS TO IDENTIFY
INFECTION WITH LYME DISEASE AND
SERIES POINTS OOLG BASED
ESPECIALLY FOR INFECTION AND
THESE ARE THE 1S WE WOULD LIKE
TO SEE THE DIFFERENCE
PARTICULARLY IN THE FIRST COUPLE
YEARS OF OUR TRIAL AND WE'RE IN
YEAR 3 RIGHT NOW, SO IT'S
PERFECT TIMING TO BE DEVELOPING
THESE THINGS.
SO JUST A QUICK REMINDER THAT
ALL BACTERIA HAVE THESE GLYCANS
AND THEY CREATE THEIR CELL WALL
IN ORDER TO KEEP THEM FROM
BURSTING THROUGH OZ METIC
PRESSURE, BUT WHAT'S SPECIAL
ABOUT BURELLIA, AS NIKI JUST
SAID AND THE SHORT PEPTIDES
WHICH DIAMEANS ARE DIFFERENT.
SO IF WE TALK ABOUT GRAM
NEGATIVE OR POSITIVE BACTERIA,
THE MOST COMMON TYPES THESE ARE
THE TYPES OF PEPTIDES YOU HAVE,
BUT FOR [INDISCERNIBLE], WE HAVE
THIS [INDISCERNIBLE] GLUE
MARIOUS SEEN FORMATION.
SO THE GOALS FOR OUR SUPPLEMENT
WERE TO SYNTHESIZE THIS
[INDISCERNIBLE] PEPTIDES AND
DR. PLATTENER AND HIS PNAS PAPER
IDENTIFIED ABOUT 13 OF THEM SO
WE TARGETED IT DOWN TO THE MOST
IMMUNOGENIC 1S BECAUSE THEY ARE
THE MOST USEFUL DIAGNOSTICALLY,
AND WE WILL CREATE THEM AS
REFERENCE STANDARDS FOR
DIAGNOSTIC PURPOSES, TO BE USED
AS INTERVENTIONS LIKE ELIZA, OR
PC R, TO LOOK AT THE ACTUAL
PRESENCE OR ABSENCE EVER THESE
PEPTIDES IN A BIOLOGICAL
SYMPTOMS, AS WE THINK PIE IN THE
SKY BIG PICTURE IF I CAN GET
KILOINGS OF IT AS IT WAS JUST
MENTIONED TO BE ABLE TO USE
THESE FOR IMMUNO LOGICAL ASSAYS,
BECAUSE WE KNOW THAT ASIDE FROM
ANTIBODY RESPONSES IT ALSO CAN
DRIVE T-CELL AND NK T-CELL
RESPONSES AND THESE ARE OF REAL
INTEREST OF MY GROUP AND
HONESTLY, IT'S A REAL GROWING
AREA IN THE FIELD OF BOTH CANCER
AND INFECT YOWZ IMMUNOLOGY.
AND THEN LASTLY, CAN YOU MAKE
THE SPECIFIC TARGETS FROM
MONOCLONAL ANTIBODY PRODUCTION
AND AND WE CAN USE THOSE
MONOCLONE'S EABT BODIES FROM
EVERYTHING FROM ARRAYS TO
SPECIFIC LATERAL FLOW ASSAYS
LIKE WHAT WE'RE USING TO
ADENTIFY SARS-COV-2.
SO AS YOU MIGHT IMAGINE THERE
WAS DELAY DUE TO PANDEMIC BUT WE
WERE SO PLEASED TO HAVE
[INDISCERNIBLE] JOIN NIKI'S
GROUP LAST YEAR.
HERE SHE IS AND MOST POST DOC, I
WAS ANAL TO GO TO IN THE NLS
JULY AND SPENT A MONTH DOING
BASIC TRAINING WITH THE GROUP TO
LEARN SO SHE WAS A MOLECULAR BI
BIOLOGIST BY TRANSLATIONAL
RESEARCH TRAINING AND TO LEARN
MORE ABOUT THE MANUEL SYNTHESIS
FOR THESE NEUROPEPTIDES I ALSO
ENJOYED DR. WILSON'S TALK
YESTERDAY AND OF COURSE NOW
BECAUSE THE GOAL IS TO HAVE AN
AUTOMATED SYNTHESIS FOR THESE
DIFFERENT PEPTIDES,
NEUROPEPTIDES.
BUT FOR NOW WE NEED TO FIGURE
OUT HOW TO MAKE THEM, THE FIRST
TIME, SO THIS IS WHAT THE GROUP
HAS BEEN WORKING ON AS WELL AS
[INDISCERNIBLE].
HERE.
AND AFTER THAT TIME, YOU KNOW
THE 3 OF THEM WOG TOGETHER TO
TRULY IDENTIFY WHAT ARE
NEUROPEPTIDES OF INTEREST AND
THE BIG 1 IS SHOWN HERE.
SO, THIS WAS IDENTIFIED BY
BRANDON AND DR. [INDISCERNIBLE]
LAB TO BE HIGH OWE IMMUNOGENIC
FOUND IN PATIENTS WITH LYME
ARTHRITIS AND BECAUSE OF THAT
THIS HYDROMIEWRIN AC IS OUR
HIGHEST TARGET IN ORD TO DETECT
IT, THEY'RE STILL FINISHING THE
SCREAMS FOR THIS IS SYNTHESIZING
IT SO WE STARTED IDENTIFYING THE
TRI PEPTIDE AS A FIRST STEP IN
MY LAB.
SO THIS WAS SENT TO US IN
FEBRUARY.
AND HOT OFF THE PRESSES, THIS IS
OUR MS TEAM AT THE UNIVERSITY OF
IOWA AND TO REMIND YOU WE DON'T
RUN MS ALL THE TIME.
WE ARE RUNNING FACTS ASHESINAL
SIS AND ELIZAS AND PERFORMING
HIGHER LEVEL EPIDEMIOLOGICAL
STATISTICAL ANALYSIS IN OUR
POPULATION, SO MS WAS NOT
SOMETHING WE NORMALLY DO AND
IT'S FANTASTIC TO SHOW YOU THIS
DATA SIMPLE THOUGH IT MIGHT BE
SO WE WERE LOOKING AT THE
MASTERY CHARGE RATIOS WHICH WERE
CONSIST WENT WHAT WE'RE
EXPECTING BRACED ON THE DATA OUT
OF THE POHL LAB AND WE WERE ABLE
TO THEN SEE THAT THAT EVEN
THOUGH WE AND LIKE--LIKE I SAID
WITH LINKAGES, WHEN WE LOOK AT
THESE PEAKS, WE CAN FIND THE TRI
PECTIDE MSMS IN HERE, AND
INDICATING THIS SPECIFICS TRI
PEPTIDE THAT IS FOUND IN
[INDISCERNIBLE], SO THE NEXT
STEPS ARE TO COMPLETE THE POETIC
TEBTIAL [INDISCERNIBLE] TARGETS,
COMBINE THEM ALL AND LOOK AT AND
FRAGMENTATION PATTERNS, THEN
WE'RE GOING TO START SOME ASSAYS
TO REALLY SEE HOW WELL THIS
WORKS AS I DIAGNOSTIC, WE WANT
TO COMPARE IT TO SAMPLES FROM
OTHER COMMON PACTERRIA THAT ARE
LIKELY TO CONTAMINATE, LIKE
E.COLI OR CANNED WHAT TO SHOW
THE FES FISCHERITY OF THIS, AND
THEN, WE'RE GOING TO GET INTO
THE ACTUAL SERUM SAMPLES FROM
OUR TRIAL.
AND BOTH FIRST WITH HUMAN SERUM
AND DIFFERENT COMPOUNDS AND THEN
GET INTO ANALYZING OUR SPECIFIC
SAMPLE SO WE'RE EXCITED ABOUT
THIS AND IT'S BEEN A REAL
PLEASURE TO WORK WITH DR. POHL.
THANK YOU ALL FOR YOUR TIME AND
ATTENTION.
NTHANK YOU VERY MUCH AND OUR
FINAL TOWK THIS MORNING BEFORE A
BRIEF BREAK IS DR. DAVID CRICH.
>> MORNING EVERYONE AND THANK
FOR LISTENING TO MY TALK AND
ALSO THANKS FOR THE NIH FOR
PUTTING TOGETHER THIS TREMENDOUS
PROGRAM.
I'M HERE REPRESENTING AND
MYSELF, WE DON'T HAVE ANY
CONFLICTS OF INTEREST THAT WE
KNOW OF.
SO, OKAY.
LET ME GO BACK 1.
OKAY, SO SO LIKE EVERYONE ELSE
WE INSPIRED BY THE PUBLICATION
OF THE NATIONAL ACADEMIES OF
SCIENCE AND WE USE THIS TO
STRUCTURE OUR PROGRAM, SO, WE
PAID PARTICULAR ATTENTION TO
THESE 2 COMMENTS HERE ABOUT THE
REPRODUCIBILITY OF GLYCOSYLATION
REACTIONS AND THE NEED TO DO
SOMETHING ABOUT THAT.
AND THEN WE ALSO PAID ATTENTION
TO THE NEED TO BRING IT INTO THE
MAIN STREAM SYNTHETIC
ORGANIZATIONS GANNAIC CHEMISTRY,
BE 1 WAY TO GO BA THIS OF COURSE
IS TO LOOK AT NEW GLYCOSYLATION
METHODS AND THAT CERTAINLY HAS
BEEN A FOCUS OF OUR UR1 PROGRAM
AND IT'S DIVIDED INTO VARIOUS
DIFFERENT SPECIFIC AIMS, SO MY
COLLABORATOR PETER GAVE A POSTER
YESTERDAY ON WHAT WE'VE BEEN
DOING WITH MICROWAVE ASSISTED
DPLI COSALATION, I'M VERY
INTERESTED IN PHOTO CHEMICAL
GLYCOSYLATION AND I--I DO THINK
THIS IS A WAY FORWARD AND I DO
HAVE NICE RESULTS THERE, BUT I'M
NOT READY TO TALK ABOUT THEM
YET.
WE WILL JUST CALL THAT A WORK IN
PROGRESS AND LEAVE IT THERE, AND
THEN, ANOTHER THEME OF OUR
PROJECT HAS BEEN THE DEVELOPMENT
OF METHODS THROUGH THE BACTERIAL
GLYCANS AND I HAVE BEEN WORKING
ON THIS FOR MANY YEARS AS WELL.
SO I WILL JUST GIVE YOU I
SNAPSHOT OF WHAT WE'VE BEEN
DOING REEBTLY SO YOU DON'T THINK
THAT I'VE GONE INTO RETIREMENT,
SO, A FEW YEARS AGO, WE GOT INTO
THE LEGION AMINIC ACID AND
PSEUDOMINNIC ACID, SO WE
DESIGNED THIS DONEAR AND
ACTUALLY WE SHOWED THAT IT
WORKED REALLY NICELY AND WITH
THE PSEUDOMENNIC ACID FOR THIS
DONOR HERE AND WE SHOWED THAT IT
WORKED EVEN BETTER SO BOTH OF
THESE CLASSES OF GLYCO SITES CAN
BE MADE NOW, THE PROBLEM IS
THATY YOU HAVE TO MAKE THE
DONORS AND THE DONOR WE USE 20
STEPS, SO WE DON'T THINK THAT
ANYONE IS ABOUT TO DO THAT
ANYTIME SOON.
SO CLEARLY WE HAVE BEEN WORKING
TO IMPROVE THIS, AND SO, THIS IS
WHAT WE PUBLISHED JUST A COUPLE
WEEKS AGO, SO WE STILL GO OUT
FROM [INDISCERNIBLE] ACID, AND
WE HAVE DESIGNED MUCH BETTER
CHEMISTRY NOW TO MAKE THE REGION
DONOR AND THE TRICK THAT WE USE
HERE BURN OFF THE SIDE CHAIN
FROM THIS TRIAL TO MAKE AN ALD
EHIDE HERE AND THEN WE USE THE
CHEMISTRY SO WE HAVE THIS WITH
12 STEPS, IT'S ACTUALLY FAIRLY
PRACTICAL AND IN ALL HONESTY,
THIS MATERIAL HERE CAN BE MADE
ON SCALE AND COULD AND SHOULD BE
SPECIALIZED.
AN YOU HAVE TO SHOOT OF THIS IS
ALSO THAT WE'RE ABLE TO MAKE
ACETA MINNIC ACID ALSO IN TWEP
STEPS AND MAKE THAT AVAILABLE,
SO WE HAVE ABOUT HALF A GRAM OF
THIS AROUND NOW AND THEN
PSEUDOMENNIC ACID, THE MOMENT WE
HAVE IT DOWN TO 15 STEPS, USING
A VARIATION ON THE SAME KIND OF
CHEMISTRY, BURN OFF THE SIDE
CHAIN AND THEN REINSTALL THE
PIECES.
THE EXTRA STEPS COME FROM THE
NEED TO PUT IN THIS AXIAL CN
BOND THERE, SO THIS CHEMISTRY IS
BECOMING PRACTICAL, WE AGAIN
HAVE SEVERAL HUNDREDS OF
MILLIGRAMS OF THIS COMPOUND.
I WANT TO COME BACK TO THIS AND
THESE STATEMENTS HERE AND I
REALLY WANT TOED TO TELL YOU
ABOUT MY LONG STANDING
PERSPECTIVE ON THIS FIELD AS A
MAIN STREAM ORGANIC KRIMMIST I
MADE SAY AND I JUST SAID MYSELF
AGAIN DESCRIBED AS PHYSICAL
ORGANIC CHEMIST IS ALWAYS A
COMPLEMENT.
EVEN AMUSING BUT I'M JUST AN
ORGANIC CHEMIST, AND MY
PERECTOMYOSINNIVE HAS ALWAYS
BEEN THAT RATIONAL ADVANCES
DERIVED FROM A BETTER
APPROXIMATE INSIGHT INTO THE
MECHANISM OF GLYCOSYLATION AND
I'VE BEEN WRITING THIS IN MY NIH
PROPOSALS CONSISTENTLY FOR 25
YEARS NOW, SO WE'VE BEEN AT THIS
FOR A WHILE AND WHERE ALL OF
THIS HAS TAKEN ME IS TO THE
STATEMENT THAT SM1 MECHANISMS
STATEMENTS NEED TO BE
DEEMPHASIZED, STRONGLY
DEEMPHASIZED YOU MIGHT SAY AND I
HAVE TALKED ABOUT THIS TO
VARIOUS REVIEW ARTICLES.
SO WE DON'T HAVE TIME TO GO INTO
ALL OF THE EVIDENCE BUT HERE'S A
GENERAL GLYCOSYLATION METHOD AND
FOR THE LONGEST TIME THE FIELD
HAVE BEEN OPERATING HERE ON THE
ASSUMPTION THAT GLYCOSYLATION
REACTIONS PROCEED VIA SM1
MECHANISMS, ON NAKED OXOCARBIN O
MIDS AND THIS IS WHERE WE GO
WRONG.
THIS IS THE CORE OF IT AND WHY
GLYCOSYLATIONS REACTIONS ARE NOT
REPRODUCIBLE.
THE REALITY IS THAT
GLYCOSYLATIONS REACT ON A SCALE
AND TO KEEP THEM THERE. TO DO
THIS WE HAVE TO HAVE THE COUNTER
IONS ARE TRIPLETS SO WOE DON'T
HAVE TIME FOR ALL THE EVIDENCE
SO WITH THE COMMON FUND PROGRAM
WE WROTE WITH MY COWORKERS 2
LARGE CHEMICAL REVIEWS THAT
BRING TOGETHER ALL OF THE
EVIDENCE AND SUMMARIZE IT, SO I
RECOMMEND THAT YOU READ THEM AND
THINK ABOUT WHAT THEY SAY.
AND THEN, IT'S NICE TO NOTE,
THAT SOME OF THE MAIN ADVANCES
IN SYNTHETIC CHEMISTRY, COMING
OUT OF THIS COMMON FUND PROGRAM
BY COMMON FUND MEMBERS, ARE ALL
HIGHLY STEREO SELECTIVE DPLI
COSALATION REACTIONS AND AT THE
END OF THE DAY, THEY ALL INVOLVE
SM2 LIKE MECHANISMS.
SO THAT AND PUSH THESE REACTIONS
TO THE EXTREMES WE DEFINITELY
NEED TO STOP THINKING ABOUT
THINGS LIKE THIS, AND WE HAVE TO
NOTED REALLY IS HOW TO SHIFT
THESE EQUILL LIBRARY FOUNDATION
RIIA FROM A BETA DONOR THAT
GIVES AN ALPHA GEICO SEED TO AN
ALPHA DONOR THAT GIVES A BETA
FLIEKOCIDE AND IF WE CAN
UNDERSTAND THAT, PROPERLY, THEN
WE CAN DESIGN HIGHLY STEREO
SELECTIVE REACTIONS, SO FOR OUR
COWORKERS WE HAVE VARIOUS
LESSONS.
PLEASE STOP [INDISCERNIBLE].
EVEN MECHANISTIC ENZYMOLOGISTS
DON'T DO THAT BUT YET WE
PERSISTED IT.
WE NEED TO THINK IN TERMS OF
CONCENTRATION AND NOTED
EQUIVALENT BECAUSE WE NEED TO
UNDERSTAND THE DIFFERENCE
BETWEEN THE RATE LAW FOR AN AN1
REACTION AND AN SN2 REACTION AND
THE DIFFERENCES IN THE WAY THEY
DEPEND ON CONCENTRATION AND IF
YOU THINK ABOUT THAT, IT TELLS
YOU THAT AS RAY ACTION
PROGRESSES AND THE
CONCENTRATIONS DIMINISH, THEN
THE MECHANISM SHIFTS OVER HERE
AND BECOMES MORE SN1 LIKE,
DURING THE COURSE OF THE
REACTION.
AND THAT'S--THAT'S AN IMPORTANT
REALIZATION.
AND THE ORDER TO COMBAT THAT, WE
SHOULD WORK ON THE FIRST ORDER
CONDITIONS AND THEN WE SORT OF
NEED TO REALLY THINK ABOUT THE
COUNTER IONS AND HOW TO PISH
THESE EQUILL LIBRARY FOUNDATION
Y RIA TO THIS SEAD OR THIS SIDE.
SO TRYING TO FORMALIZE ALL THIS
AND GET THIS MESSAGE ACROSS TO A
BROAD AUDIENCE, PETER AND I WE
PUT TOGETHER THESE FIED LINES
FOR OLD GRIEK O SIDE FORMATION
NAFIRST PRINCIPLES THAT WE
PUSHLISHED LAST YEAR IN ACS
CENTRAL SCIENCE.
SO CAN YOU SEE THAT WE PUT
TOGETHER 12 GUIDELINES FOR DOING
SEROSELECTIVE GLYCOSYLATION AND
THE MAIN FEATURES ARE
CONCENTRATION, TEMPERATURE, AND
THE COUNTER ION AND THESE
GUIDELINES WERE DEVELOPED TO
DIFFERENT PLACES ON I75 OR IN
PROXIMITY TO IT, AND TO KEEP THE
TEMPERATURE DOWN, OF THE YIELD
OF THE SELECTIVITY AND TAKE NOTE
OF THE COUNTERION.
THOSE ARE THE CORE MESSAGES, SO,
GUIDELINE 1 IS FAIRLY OBVIOUS,
WE NEED TO KEEP GOOD
EXPERIMENTAL BOTS, AND IN
PARTICULAR, WE NEED TO DISCUSS
THE CONCENTRATION AND THE AMOUNT
OF SOLVENT THAT WE USE.
THEN, IN ORDER TO DO
COMPARISONS, WE SHOULD--WE
SHOULD NOTE THAT WE SHOULD--WE
SHOULD PICK A STANDARD
CONVERSION OR PRODUCT YIELD, WE
SUGGEST AN 80% YIELD AND IF
EVERYONE REPORTED REACTIONS AT
THE SAME CONVERSION, IT WOULD
MAKE COMPARISON BETWEEN
DIFFERENT METHODS MUCH EASIER
AND MUCH MORE ACCURATE.
BECAUSE OF THE CONCENTRATION
PROBLEM WE SHOULD WORK UNDER THE
PSEUDOFIRST ORDER CONDITIONS AND
BIG AT THAT TIME DAILY BASIS
APPROACHES TO MAKING PREDICTIONS
SHOULD ONLY DUCE THAT DATA THAT
HAS THE CONCENTRATION IN IT.
A SECOND IMPORTANT THING IS
TEMPERATURE.
AGAIN IF YOU THINK OF THE
DIFFERENT ENTROPY REQUIREMENTS
FOR THE TRANSITION STATE OF AN
SN 1, AND AN SN2 REACTION, IT'S
OBVIOUS THAT IF YOU ALLOW THE
TEMPERATURE TO CHANGE DURING THE
COURSE OF A REACTION, THE
MECHANISM WILL CHANGE.
SO WHAT WE NEED TO DO IS TO
OPERATE OUR REACTIONS AT A
CONSTANT TEMPERATURE AND THAT IF
WE WANT TO DO THE SN2 LIKE
MECHANISMS, THEN, THE REACTION
SHOULD BE CONDUCTED AT THE
LOWEST POSSIBLE TEMPERATURE 1
CYST WENT OUR PRACTICAL'S ACTION
TIME AND THEN WE HAVE TO THINK
ABOUT COUNTER IONS AND SO ON AND
SO FORTH.
AND IT SO HAPPENS THAT
[INDISCERNIBLE] IS A GOOD
COUNTER ION, --IF FACT WE HAD TO
TALK ABOUT THE ACCEPTAR AND
MAKING IT MORE NUCLEOPHILIC AND
PROTECTING GROUPS AND SO ON,
THEY LEAD TO COMPLICATIONS AND
THEN, WE SHOULD WORRY ABOUT
SOLVENT AND DISCIPLINARY CLOOR
MEAN IS ULTIMATE, SO WE
PUBLISHED THESE, IN THE HOPE
THAT PEOPLE WILL READ THEM AND
REALLY THINK ABOUT THEY'RE DOING
DPLI COSALATION REACTIONS AND WE
THINK IF YOU FOLLOW THIS LIST OF
GUIDELINES THAT THEN, WE SHOULD
BE ON THE WAY TO MAKING THIS
FIELD MORE REPRODUCIBLE, WHICH
WOULD BE AN ACHIEVEMENT, SO THE
QUESTION AND THE LAST SLIDE IS
ABOUT THE NEXT STEPS AND WHERE
DO WE GO FROM HERE.
AND WHAT I WOULD REALLY LIKE FOR
US TO DO NOT JUST MY LAB BUT AS
A COMMUNITY IS TO BUILD ON THE
GIED THAT WE PUBLISHED AND BUILD
STANDARDS ON THE DPLI
COSALATION'S ACTIONS SO THAT
PEOPLE CAN REPRODUCE THEM
THOUGHTFUL AND THEN FOR MAKING
COMPARISONS BETWEEN METHODS, SO
THAT WE CAN--WE CAN REALLY
DECIDE WHICH 1 IS BEST, AND
WHICH METHOD WE SHOULD ALL
FOLLOW.
SO, THOSE ARE THE KIND OF THINGS
THAT PETER AND I ARE INTERESTED
IN.
AND CLEARLY THAT'S A WORK IN
PROGRESS AND I'M LOOKING FORWARD
TO THE FEEDBACK FROM OUR
SYNTHETIC COLLEAGUES, HOPEFULLY
AT LUNCH TIME AS TO HOW WE MIGHT
GO FORWARD WITH THAT.
SO, THIS WORK HAS BEEN DONE IN
GEORGIA AND IN TO LEEING LIDO.
CLEARLY WE SHOULD THANK DAVID
WALT AND THE AUTHORS OF THE NRC
REPORT.
AGAIN, PAM MARINO AND THE GRANT
AND MANY COWORKERS OVER MANY
YEARS FOR THE CONTRIBUTION.
SO THANK YOU.
[ APPLAUSE ]
>> SO 1 THING TO BE AWARE OF IS
THAT FLOW CHEMIST RADIOY ALLOWS
TO YOU DO CONSTANT TEMPERATURE,
SO THAT'S WHY WE'RE EXCITED
ABOUT LOOKING AT THOSE OF THOSE
REACS LOOKING AT THESE
CONDITIONS, THIS ENDS THE EARLY
MORNING PROGRAM AND NOW I
TURNOVER THE FLOOR TO AMANDA.
I JUST WANT TO MAKE A FEW QUICK
ANNOUNCEMENTS, WE ARE GOING TO
SKIP THE BREAK, SO IF YOU NEED A
BREAK, LEAVE, COME BACK IN.
BLAWZ WE'RE RUNNING LATE.
WE WILL RUN INTO THE NEXT
SESSION CHAIRED BY DR. TMYER BUT
JUST A FEW OTHER ANNOUNCEMENTS
IF YOU EVAPORATE PICKED UP YOUR
BREAKFAST, PICK UP YOUR
BREAKFAST AND MAKURE YOU PAY PAM
FOR BREAKFAST, LUNCH AND DINNER
IF YOU ORDERED ANY OF THOSE AND
IF YOU ARE IN THE AFTERNOON
SESSION, AT 12:15 AS SOON AS
THIS SESSION ENDS PLEASE COME UP
AND UPLOAD YOUR SLIDE FIST YOU
EVAPORATE DONE SO YET.
THANK YOU.
>> --DR. CHEN, 60 SIS AND
REPLICATION OF GLYCO LIPIDS.
>> THANK YOU FOR THE
INTRODUCTION, AND I WOULD LIKE
TO THANK OTHER ORGANIZERS FOR
PUTTING THIS MEETING TOGETHER.
IT'S ALWAYS A GREAT PLEASURE TO
BE HERE IN PERSON.
AND TODAY, I AM GOING TO SHARE
WITH YOU OUR CONTRIBUTION FOR
THE DEVELOPMENT OF METHODS,
REGARDING WITH THE FOCUS ON
MEDICARE
MAMMALIAN LIPIDS.
HAD IS A COLLABORATION WITH MY
GROUP AT UC DAVIS AND THE
PROFESSOR GROUP AT THE GEORGIA
STATE UNIVERSITY.
AND THIS WAS A COLLABORATION
EFFORT THAT IS FUNDED BY UO1
COMMON FUND FLIEK O SCIENCE
PROGRAM.
ANDUREENTLY WITH WORKING WITH
IMCS IN TRANSLATING OUR P1S, THE
STRATEGIES AND THE KIDS FOR
ACCESSING FLIEK O SINGLE LIPPITY
EDUCATIONALS BY COMMERCIALIZING
THEM--HOWEVER, DETAILS OF THIS
MECHANISM IS NOT CLEAR.
THE MAJOR REASONS IS THE LACKING
OF FOR EXCESS TO STRUCTURALLY
DEFINED GLUE MARIOUS COSSINGLE
LIPIDS.
AND IN ADDITION TO SERVING
EXTENDERS, AND THE PROBES FOR
RESEARCH, LIPIDS ARE POTENTIAL
THERAPEUTIC REAGENTS ESPECIALLY
GREAT POTENTIAL FOR TREATING NEW
ROLE DAMAGES AND THE NEUROLOGY
CODE DISEASES SUCH AS HUNTING
TONS AND THE PARKINSON'S
DISEASES AND THERE'S A URGENT
NEED TO ACCESS THESE
STRUCTURALLY DEFINED GLYCO
SINGLE LIPIDS IF LARGE AMOUNT
ESPECIALLY IF YOU WANT TO USE
THEM AS A THERAPEUTIC REAGENTS
AND LET'S TAKE A LOOK AT THE
COMMERCIALLY AVAILABLE SOURCES.
ACTUALLY THOSE ARE VERY LIMITED.
AS YOU CAN SEE FROM THE
[INDISCERNIBLE] POLEAR LIPIDS,
THIS SIMPLE [INDISCERNIBLE]
DENDRITIC CELLROCYTE, THE SPRIES
EXTREMELY HIGH.
AND THE MORE PROBLEMATICALLY,
THE AVAILABLE REAGENT ACTUALLY
IS A MIXTURE AS THE POINT OUT IN
THE WEBSITE, THIS ONLY IS THE 1
OF THE STRUCTURES THAT SO AMINE
SYSTEM VERY HARD TO ACCESS
STRUCKURALLY DEFINED GLYCO
SINGLE LIPIDS WITH DEFINED GLYCO
COMPONENT AND THE LIPID
COMPONENT.
AND MAMMALIAN LIPIDS CAN BE
MOVED INTO 5 GROUPS.
ALL OF THEM ACTUALLY HAS THIS
COMMON [INDISCERNIBLE] ABILITY
COMPONENT WHICH IS CALLED ACET
MID.
THIS HAS SINGLE STRAND COMBONENT
WHICH IS FOUND WITH THE FATTY
ACID, BOTH OF THESE COMPONENTS
CAN BE DIFFERENT.
SO FOR THE SINGLE CELL PART, IN
NATURE YOU CAN 28 OR 18
[INDISCERNIBLE].
FOLD UP THE ASSAY FOR THE
STRUCTURE OF VARIOUS PREMELY
CANNED WHAT DIFFERENT ON THE
SIZE AND ALSO DEGREE OF
SATURATION AND ALSO STRUCTURAL
MODIFICATIONS.
ALSO WITH THE LIPIDS ACTUALLY
SHARE A COMMON CORE WITH LACTOSE
THAT IS BETA TO THE CELL
[INDISCERNIBLE].
AND THIS CELL BY ITSELF IS
INSOLUBLE IN WATER.
HOWEVER, IF YOU REMEEF THIS
APART.
THE LACTOSE SINGLE ASSAY ITSELF,
IT'S A RELATIVELY--READILY
SOLUBLE IN WATER, SO IT'S A VERY
SUITABLE ACCEPTOR SUBJECT FOR
GLUE MARIOUSICOSAL TRANSFER ACE
CATALYZE REACTION THAT CARRIED
OUT IN THE SOLUTION.
AND ALSO THE NATURAL OCCURRING
[INDISCERNIBLE] COMPONENT
PROVIDES A VERY GOOD TECH,
HYDROPHOBIC TECH TO ALIEU EITHER
PURIFICATION OF THE PRODUCTS, SO
OUR GOAL TO SET UP AT THE
BEGINNING OF THE YEAR WAS TO
DEVELOP PURIFICATION STRATEGIES
FOR FLIEK O SINGLE LIPIDS AND
TO,A LOW EVEN NONSPECIALISTS CAN
ACCESS THIS KIND OF COMPOUNDS
WITH VERY CONVENIENT TOOL BOX
AND ALSO VERY EASY PROCEDURES.
SO WE BASE THIS ON IMPORTANT
INTERMEDIATE LACTOSE SELLS WHERE
WE GIVE UP OUR STRATEGIES IN 3
STEPS.
THE FIRST STEP BASICALLY IS BY
CHEMICAL SYNTHESIS, TO ACCESS
MODULE AMOUNT OF LACTOSE SINGLE
STRAND AND THEN, STEP 2, IS TO
DO THE ENZYMATIC EXTENSION OF
THE GLYCO COMPONENT OF AND THIS
ACTUALLY IS THE BASED ON
THEENTIOUS FICIENCY OF THIS
GLYCO TRANSFER ACE CONTAINING 1
PARTICLE ENZYME SYSTEM, WITH THE
FACILITATION OF THE SINGLES IN
COMPONENT FOR PURIFICATION.
SOME WE HAVE OBTAINED THE GLYCO
[INDISCERNIBLE] INTERMEDIATES,
BY A SINGLE PURIFICATION IN LESS
THAN NIRT MINUTES AND STEP 3,
BASICALLY IS DOING THE ISOLATION
WHICH IS VERYENTIOUS FICIENT
STRATEGY TO INSTALL DIFFERENT
KIND OF FATTY TRAIN IN THE LAST
STEP TO PROVIDE A WIDE VARIETY
OF THE TARGET STRUCTURES.
SO STEP 1, ACTUALLY WEIGHT AND
GRABBED THE 4 GENERATIONS OF THE
SYNTHETIC STRATEGIES.
THE FIRST GENERATION WAS ALWAYS
THE SINGLE SCENE, WE HAVE 8
STEPS OF THE TRANSFORMATION, WE
OBTAINED THIS IMPORTANT TPHREU
RACING CO ACCEPTOR THAT ALLOW US
TO DO THE DPLI COSALATION
REACTION WITH LACTOSE DERIVED
DONOR.
WE WERE AICIAL TO DO THIS
REACTION IN LARGER SKILL AND
WITH RELATIVELY HIGH YIELDS.
THE LIMITATION ACTUALLY IS THAT
WE CAN ONLY OBTAIN THE LACTIC
ACIDOSEISS ON [INDISCERNIBLE]
AND THE OTHER SPINNING O SWEEN,
WITH THE 18 COVERS SPHINGOSIGN,
AND THIS START WITH THE LESS
EXPENSIVE L-SERIES POINTSINE
BUILDING BLOCK.
AND WE'RE ABLE TO ACHIEVE ALSO
MORE THAN 10 GRAND SCALE SYNTH
AND THE FOURTH GENERATION OF THE
SYNTHETIC STEPS AND THIS
ACTUALLY ALLOWS TO ACCESS THE
TARGET IN LESS STEPS AND THE
ALSO WITH MUCH LOWER COST AND
THIS WILL ALLOW US TO BE ABLE TO
PRODUCE THE LACTOSE SPINGOSIGN
IN LESS EXPENSIVE COST.
SO WITH THE LACTOSE AND--AND AND
THE DIVERSE OF THE TARGETS.
AND EVERY STEP ACTUALLY SAY
PURIFICATION ALLOW US TO GET THE
TARGET IN LESS THAN 30 MINUTES.
AND I ALSO WANT TO HIGHLIGHT THE
ADVANTAGE OF THE ENZYME SYSTEM
ALLOWS US TO INTRODUCE MODIFIED
SUGAR STRUCTURES, INTO THE
TARGETS.
SO OVERALL 15, 1 PART
[INDISCERNIBLE] SYSTEMS CAN BE
USED TO GENERATE MOST OF THE
GLYCO LIPID TARGETS AND I WILL
HIGHLIGHT 4 OF THEM WHICH CAN BE
USED TO SYNTHESIZE MAJORITY OF
THE IMPORTANT CELLULAR GLYCO
[INDISCERNIBLE].
AND OF THEM, ACTUALLY START WITH
SIMPLE MONOSACCHARIDES WHICH ARE
INEXPENSIVE AND COMMERCIALLY
AVAILABLE AND THEY CAN BE
ACTIVATED TO GENERATE SUGAR
NUCLEOTIDE THAT CAN BE USED UP
BY THE SUITABLE TRANSFER ACE TO
MAKE THE THESE OTHER TARGETS.
AGAIN THE COMPONENT IN THE
PRODUCT HELPS FOR THE
PURIFICATIONS PROCESS THAT CAN
BE DONE IN LESS THAN 30 MINUTES,
USE THE SYMPOSIUM
[INDISCERNIBLE].
AND AS A THUMB POST HAS
SUCCESSFULLY SYNTHESIZED FOR
GANGLIA BASED PRIORITIZED CANCER
AND THE GENES WERE COMPLETED
WITH 1 OR MORE REACTIONS WITH
VARIOUS SKILLS AND FOR THE
[INDISCERNIBLE], THE INDIVIDUAL
TO MAKE MORE THAN 1 GRAM IN THIS
PRODUCT AND AFTER STEPS ACTUALLY
OBTAINED IN HIGH YIELDS,
APPROXIMATE THE USE ISOLATION
CONDITION THAT PURIFY THE
PRODUCT VERY EFFICIENTLY.
AND WE ALSO ALSO INTRODUCE
MODIFY SUGARS TO THE FINAL
PRODUCT AS A DRUM HOLE, THERE'S
ALWAYS THIS [INDISCERNIBLE]
ASSAY, AND USE SALIC ACID,
OTHERWISE BASE THE REACTION TO
GENERATE THIS GM-3 CONTAINING
DIFFERENT SIALOGLYCANSIC ACID
FORMS.
AND CURRENTLY WE ARE WORKING
WITH THIS TO FURTHER IMPROVE THE
STABILITY, EXPRESSION LEVELS OF
THE KEY FLIEK O TRANSFER ACES
THAT ARE INVOLVED IN THIS
SYNTHETIC PROCESS, BY APROTEIN
ENGINEERING PROCESS AND ALSO
WITH IMCS EXPERTS.
ON THE FRAGMENTATION PROCEDURES.
AND WE ALSO FURTHER IMPROVE THE
PROCESS, BY IMPROVING CHEMICAL
SYNTHESIS OF THE LACTOSE
[INDISCERNIBLE] AS WELL AS TO
IMPROVE THE OPMI AND THE MEDICAL
PROCESS, IF WE HAVE OUR TARGET
IN MIND, ACTUALLY WE DON'T NEED
TO PURIFY THE INTERMEDIATE
PRODUCTS AND BY THIS WAY, WE CAN
MINIMIZE THE PURIFICATIONS STEPS
AND ALSO WE FURTHER IMPROVE THE
PROCESS BY CHANGING THE
CONDITION AND THE DECREASED
AMOUNT OF THE ENZYME THAT CAN BE
USED AND WITH SUCCESS RATE, WE
DID THIS, GRAM SKILL SYNTHESIS
OF THE GM 1 AND WE ARE WORKING
WITH MATERIALS MAKING THE
10-GRAM SCALE AND MAYBE EVEN
LARGER SCALE OF THAT AND WITH
THOSE TARGET FLIEK O
[INDISCERNIBLE] IN HAND WE CAN
DO THE FINAL ISOLATION REACTION
AND A COMPELLED TO OUR EARLIER
CONDITIONS WHICH WASSA, CHIEFED
25% YIELD IN 84 HOURS STARTING
WITH THE FATTY ASSAY AS 1 OF THE
STUDY MATERIAL.
WE FOUND OUT THE ACL
[INDISCERNIBLE] IS A BETTER
STUDY MATERIAL AND THE REACTION
CAN BE COMPLETED IN 2 HOURS
IEWOF THE JUSTUTESSING THE
COGNATE IN [INDISCERNIBLE].
AND WE HAVE VERY HIGH YIELDS FOR
THE TARGETS AND AS EXAMPLE, WE
CAN STORE DIFFERENT SIZES.
AND DIFFERENT DEGREE OF
SATTURATION OF THE FATTY ACID
SAY ON TO THE FINAL PRODUCT.
SO BASICALLY ALL THIS WERE
ACHIEVED IN VERY HIGH YIELDS.
SO IN SUMMARY, WE HAVE ACHIEVED
OUR NUMBER 1 GOAL AND OUR RESULT
VS BEEN SHARED WITH THE
COMMUNITY BY RESEARCH PAPERS, WE
ARE ALSO STILL IN THE PROCESS OF
REPAIRING NEW PAPERS WHICH WILL
COME OUT LATER IN YEAR AND THE
PROTOCOL FOR THE SIPGHT SIS OF
THE GD3 AND ALSO GM3, COVERAGED
IN THIS GLYCO SCIENCE SPECIAL
COLLECTION ORGANIZED BY
[INDISCERNIBLE] AND ALSO
[INDISCERNIBLE].
AND WE ALSO SHOW OUR RESULTS IN
TALKS AND THE THE CROSS
VALIDATION WAS DONE BY THE TEAM.
AND WE ALSO SHARE OUR ENZYMES
WITH SEVERAL GROUPS AND FOR MORE
DETAILED INFORMATION PLEASE
CHECK OUT THE WEBSITE THAT
DR. [INDISCERNIBLE] AT GEORGIA
STATE UNIVERSITY ESTABLISHED.
AND WITH THAT I WOULD LIKE TO
THANK OUR TEAM MEMBERS WHO
CONTRIBUTE TO THE WORK THAT ARE
PRESENTING AND ALSO OUR
COLLABORATORS AND THE GROUP FOR
CROSS VALIDATION EFFORT AND OF
COURSE I WOULD LIKE TO THANK THE
FINANCIAL SUPPORT FROM NIH
COMMON FUND GLYCO SCIENCE
PROGRAM AND ALSO NIGMS WITHOUT
THIS FINANCIAL SUPPORT, OUR
EFFORTS IN ADVANCING THE GLYCO
SCIENCE FIELD WILL NOT BE
POSSIBLE.
AND WITH THAT, IRB WOULD LIKE TO
THANK YOU FOR YOUR ATTENTION.
[ APPLAUSE ]
, THANKS.
>> OUR NEXT TALK IS FROM DR. YU
FROM UNIVERSITY CALIFORNIA DAVIS
AS WE RICOCHET BACK AND FORTH
BETWEEN POLYSACCHARIDES AND
LIPIDS.
FOR CHEMOEN ENZYMATIC SYNTHESIS
OF BACTERIAL POLYSACCHARIDES.
>> THANK YOU MICHAEL.
SO OUR U01 PROJECT IS ABOUT
CHEMO ENSWRANSS OF
POLYSACCHARIDE.
WE ARE CLOBERATING WITH LAURA AT
M. I.T. AND [INDISCERNIBLE] AT
GEORGIA STATE.
THE INTEREST CURRENTLY SVR, WITH
THE [INDISCERNIBLE] AND
COLLABORATE WITH [INDISCERNIBLE]
FOCUSING GENES SIPGHTICIZING
DPLI KAN--KANAS.
WE KNOW THIS IS A MAJOR PROBLEM,
BY THE SURVASE, IT'S A PACTERIAL
POLYSACCHARIDE.
SO THE POLYSACCHARIDE ON THE
BACTERIAL VECTOR, THE OTHER
VACCINE TARGET AND THE CAUSE OF
THE TYROSINE CANDIDATE, SO
DETECTED BY THE POLYSACCHARIDE
IS VERY POTENT.
SO THE PROBE TO STED THE RELATED
INTER--PROBE TO STUDY THE
RELATED INTERACTIONS AND THE
MATERIALS AND IMPROVE THE
PROTEIN THEOR PUTTICS.
SO ALSO AWARE OF THE IMPORTANT
PROBE TO STUDY THE ARBO HYDRATE
ENZYME HOWEVER CURRENTLY MANY OF
THE LABS SOMEHOW ARE AVAILABLE.
THE 3 TO PRODUCING
POLYSACCHARIDE IN THIS GROUP,
MANY ARE FOCUSING ON THE
SYNTHESIZING [INDISCERNIBLE]
DEPENDENT POLYSACCHARIDE TARGET.
LAURA IN OUR GROUP TRYING TO
[INDISCERNIBLE] INDEPENDENT
TARGET.
HOWEVER, WE PARTICULARLY
[INDISCERNIBLE] PALY SACCHARIDE,
INCLUDING THE [INDISCERNIBLE],
DEFINE, AND [INDISCERNIBLE].
BACTERIA CAN CAUSE LIFE
[INDISCERNIBLE] MOTIFS.
SO THE BACTERIAL POINT HAS A
SEPARATE UNIT, WHICH IS BY THE
SYNTHESIS.
THE PROBLEM WITH THE SYNTHESIS
IS THAT THEY HAVE THE
[INDISCERNIBLE] PENN STATE FOR
THE IMPLAY 1 PORT TO THE ENZYME
FOR THE DPLI COSALATION COMBINED
WITH THE BACTERIAL 60 SIS TO
PRODUCE DPLI KAN--KANAS, IN THE
[INDISCERNIBLE] NUCLEOTIDE, AND
FROM THE SIMPLE MONOSACCHARIDE,
TRYING TO BY THE BACTERIAL
SYNTHESIS,OT 1 PAGE THE OPM E IS
AWARD USING [INDISCERNIBLE],
NUCLEOTIDE, AND THE NEED IT TO
INCORNERATE THE MODIFIED SUGAR
TO THE DPLI KAN--KANA SUCH AS
[INDISCERNIBLE] AND IKAN--KANA
GROUP TO ALLOW FOR THE
MODIFICATIONS.
SO BY EXAMPLE, THE OPME, WILL
ASSIMMULATE IN AN DROJ NOWS
[INDISCERNIBLE] POLYSACCHARIDE
DPLI KAN--KANA.
THE DISCIPLINARY SACCHARIDE,
SINCE THIS IS CALLED
[INDISCERNIBLE] BETTER FUNCTION
FOR THE ENZYME, YOU JUST KILLED
THE POLYSACCHARIDE AND YOU
UPROOT WE START ON THE
[INDISCERNIBLE] FOR THE NEW OOH
C, AND AND DPLI COSALATION IN
THE [INDISCERNIBLE] TO FORM THE
[INDISCERNIBLE].
CAN SHOW THE GRAND SCALE
INCIDENT.
THE AFULLATION FROM THE
[INDISCERNIBLE].
THE CPT LAB TO PURIFY THE
PRODUCT, USING THE IT CAN BE
EASILY REMOVED--GLYCOSYLATION,
IT'S A DIFFERENT SITE TO THE
SACCHARIDE.
UPON IT WAS ABLE TO STUDY THE
DIFFERENT SHAPE BETWEEN THE SET
OF THE SUBSET, AND THE
[INDISCERNIBLE].
IT FUNDED THE GUARANTEE OF THE
[INDISCERNIBLE] WHEN YOU CAN
[INDISCERNIBLE] THE CENTER WILL
NOT AFFECT THE DOSE
[INDISCERNIBLE].
SO FURTHER, THE [INDISCERNIBLE]
OF THIS [INDISCERNIBLE] ENZYME
VERY BROAD CONOR SPECIFICITY BUT
IT CAN TOLERATE A LOT OF
MODIFICATIONS ON THIS,
ESPECIALLY THE IT CAN TAKE THE
[INDISCERNIBLE], AND THE CMP,
ACID TOLERANT, THE PROPERTIES
USING THE [INDISCERNIBLE] OR THE
SIXTH [INDISCERNIBLE].
ADDING SALIC ACID SEPRESSOR FROM
STARTING FROM THE SACCHARIDE,A
PLIES OPME, STARTING THE CANNED
WHAT AND RADIATION PRODUCE
LIBRARY FROM THE TRI SACCHARIDE,
TO UNDER THE 10 TO THE
SACCHARIDE, AND WHICH YOU SHOULD
THE STRICT ASSAY, THOSE ARE
SEVENTH OF THE NINTH
MODIFICATION, SO, IT CAN BE
READILY [INDISCERNIBLE] AS A
GROUP SO THOSE TPHREU RACING
KAN--KANA FLOWED AS A STABLE
PROBE AS THE TPHREU RACING
KAN--KANAS.
HAVE D AND THE K5 POLYSACCHARIDE
AND THE 2 BART ARE THE SAME,
THEY SEPARATE THE UNIT AND THE
PM IT SHOWED 2 IS THE BI
FUNCTIONAL ENZYME FOR THE YOUTH
TO SYNTHESIZE THESE
POLYSACCHARIDE.
AND ON THE TRUNCATED DATE,
8:00 P.M. TO 2 TO HOW TO IMPROVE
THE SUPPRESS AND HOW BETTER THE
STABILITY IS TRUNCATED IN THE
SENTENCES.
SO APPLYING THE OPME, AND THE
LOCAL TRANSFERAISES, START FROM
THE SMALL MONOSACCHARIDE WE
SYNTHESIZED UP TO
[INDISCERNIBLE] SACCHARIDE, SO
EACH PROGRAM WAS PRODUCED ON
SCALE.
SO WHEN TRYING SETTING, THE
GLYCAN SUCH AS A PHARMA
[INDISCERNIBLE] TO HAVE THAT
SACCHARIDE, FOR THE FIRST
CAUSEIDATION FOR THE ENZYME WAS
THERE, WHICH MEANS WHEN YOU TRY
TO USING THE SYNTHESIZE, BESIDES
THE HYDROCELLS ARE THE DIFFERENT
SET OF [INDISCERNIBLE]
[INDISCERNIBLE] TO OVERCOME THIS
[INDISCERNIBLE] WITH THE 2
MUTANTS.
ONE MUTANT, 1 SINGLE FUNCTION OF
ATT, AND HOW IT LOOKS NEGATIVE 2
TWORKS T.
AND TO TURN THIS OUT, THERE ARE
DPLI KAN--KANAS SO WE WERE ABLE
TO SEND THIS IN, OUT OF THE
SACCHARIDE, WITH THE YIELD.
OKAY, [INDISCERNIBLE] AND THE
CHEMO [INDISCERNIBLE] YOU START
FROM THE UDP SERUM WITH INSITUE
[INDISCERNIBLE] BY UJM, WHICH
GENERATE A 4 SIDE
[INDISCERNIBLE] OF THE
[INDISCERNIBLE] MUTATION,
INTRODUCING AS A HANDLE ON THE
TERMINAL SUGAR.
SO PRODUCT CAN BE READY TO
PURIFY, IN THE SOLUMKC.
AND HE ALSO FUNDED THE GTAC,
THIS CAN TAKE THE MODIFIED SUGAR
LIPID AS A DONOR AND YOU
COMPUEITATE, INCORNERATE IT'S
MODIFIED RABBINOSE WHICH WOULD
ALLOW THE FROZEN [INDISCERNIBLE]
BY THE CHEMISTRY.
THE 3 DIFFERENT SUGAR
[INDISCERNIBLE] WHICH IS THEOT
CARBON STRAIGHT, CARBON 5 AND
FOUND OUT THE DIFFERENT PROBE
CAN BE PERFERED BY THE DIFFERENT
PATIENTS FOR EXAMPLE,
[INDISCERNIBLE], OF THE PA, AND
[INDISCERNIBLE] SO THE PROBES,
HOW MANY ARE USED FOR
PUBLICATIONS FOR THIS CAN BE
USED FOR THE HYGIENES OF THE
DPLI CAN IN THE SEMESTER WALL,
MORE COMPLEX SYSTEM, FIRST THE
[INDISCERNIBLE], YOU INCORPORATE
THE 2 [INDISCERNIBLE] RABBINOSE
INTO THE TPHREU RACING KAN--KANA
OR THE [INDISCERNIBLE].
WHEN TD TARGET GOES TO THE
MACROPHAGE CAN BE OBSERVED, YOU
CAN THAT FOR THE PROBE TO
MONITOR THE CHANGING, DURING THE
[INDISCERNIBLE].
THIS GROUP TARGETED AND
[INDISCERNIBLE] 71 DEPENDENT
TARGET FOR EXAMPLE, THIS IS PF2,
AND SIDE WHAT 3 DEFINITE
POLYSACCHARIDE, SO IT'S A P A S
2, IS THE MOST ABUNDANT 1 WHICH
HAS THE HETEROCANNED WHAT.
THE HOW ALL THE [INDISCERNIBLE]
TO TRANSFER ACE, THAT STAND FROM
THE CHEMICAL INSIDE THE
MONOCELLULAR REPAID, IT PRODUCE
THIS NEOINATE ON THE SCALE.
THE GROUP ALSO CO INSIDE A
NUMBER OF THE [INDISCERNIBLE].
THE [INDISCERNIBLE] ORGAN SAY
WHY, [INDISCERNIBLE] NEOINATE.
WE KNOW THIS IS A POLYMERASE
[INDISCERNIBLE], IS HARD TO DO
THE SKILL.
THIS GROUP IS VERY DEFICIENT IN
THE CHEMICAL APPROACH TO COUPLE
THE NEOINATE FORM THE
[INDISCERNIBLE] FOR OUR
ENGAGEMENT.
THE [INDISCERNIBLE] OR THE 1
PAGE THAT WE HAVE THE
HYDROLYSIS.
SO THIS APPROACHED BY THE
HYDROXIDE, SUGAR PLAT, THIS IS
ANOTHER SUGAR CONTAINED THE
GROUP.
THE REST OF THE WATER CAN BE
FINISHING IN HALF AN HOUR, AND
THE GROUP HAS IS A LAB
COLLABORATOR AND TEND TO SUFFER,
USE THIS APPROACH,
[INDISCERNIBLE] PROTEIN NEOINATE
WHICH IS A START FROM
THE--COUPLE THE TRI SACCHARIDE
WITH THE FLOW RATE.
[INDISCERNIBLE] DROKSIDE WITH
THE OTHER GROUP USING MUTATIONAL
2 ENZYME WITH 2 SUGARS, FOR THIS
NEOINATE, CURRENTLY IN THIS
GROUP IS WORKING OUT, YOU USE
THE SAME STRATEGIC PLAN EMGY TO
REACHING MUCH LONGER REPEATING
NEOINATE, AFTER 20.
OKAY, SO CURRENTLY WE ARE
WORKING ON THE PROTOCOL, AND WE
ARE DEVELOPMENT OUT IN THIS--FOR
THIS WEBSITE, AND FOR OTHER
PROTOCOL READY ANDY ARE ALSO
PRODUCING [INDISCERNIBLE].
WHILE WORKING WITH PROFESSOR
[INDISCERNIBLE] AT UCD, AND TO
PRODUCE THE BIOMATERIALS, SINCE
THAT MATERIAL DPLI KAN--KANAS
AND OUR [INDISCERNIBLE].
IS DOING THE CROSS VALIDATION,
ALSO CROSS VALIDATION, AND ALSO
TO PROTECT OKAY, FINALLY, I WILL
THANK ALL OF OUR TEAM MEMBERS,
ON THE THANK YOU FOR THIS WORK.
THANK YOU FOR YOUR PATIENCE.
[ APPLAUSE ]
>> THE NEXT SPEAKER WILL BE
ANDREW LEE FROM IMCS.
>> TESTING OKAY, THANK YOU, I AM
ANDREW LEE AND I THANK THE
ORGANIZER AND PAM FOR INVITING
ME HERE, IT'S MY FIRST TIME
PRESENTING IN FRONT OF THIS
AUDIENCE, I WILL CONFESS, I HAVE
NO GLYCO EXPERIENCE WHATSOEVER
AND WHAT AM I DOING HERE BUT
WE'LL ACTUALLY START WITH 1
SLIDE BACK WE ARE COLLABORATING
AND WE DID A FANTASTIC
INTRODUCTION, WE ARE PART OF
THAT ENZYME SCALE UP COMMERCIAL
ENTITY.
SO THE TITLE SAYS, TRANSITIONING
IN THE BENCH SCALE BACK TO
INDUSTRY SCALE FROM YOUR HANDS
BACK TO THE PENCH.
WHAT IMCS DOES IS WE DESIGN,
DEVELOP AND MANUFACTURE AND
DISTRIBUTE.
SO FROM THE START TO FINISH, OF
ALL ENZYME PRODUCTS AS WELL AS
THE CHROMATOGRAPHY PRODUCT.
AND IF YOU HAPPEN TO CONFUSE THE
ACRONYM, IMCS, THINK OF IT AS AI
AM A CUTE SCIENTIST, JUST
REMEMBER TO THE ACRONYM.
SO IMCS IS A PRETTY YOUNG
COMPANY.
WE STARTED IN 2013 AND WE
ACTUALLY FOCUSED ON
MICROCHROMATOGRAPHY PRODUCTS,
BUT WITHIN 6 MONTHS WE KICKED
OFF AND FOUND A BETA GLUE
CHRONODACE THAT TOOK OVER THE
URINE DRUG TESTING INDUSTRY.
SOPHISTICATEDY IN AMERICA IF YOU
KNOW PRESIDENT OPIOID CRISIS AND
YOU'RE IN DRUG TESTING IT IS
JUST AOF THED ROUGH ATOM
NATIONAL LIBRARY OF MEDICINEICAL
NUMBERS SO THIS ENZYME REPLACED
THE WHOLE URINE DRUG TESTING
MARKETS AND WE SCALE UP PRETTY
RAPIDLY SO WE'RE PRETTY FAMILIAR
WITH THE SAIL UP ENZYMES SO IN
TYPICAL ENZYME PRODO YOU
RECOGNIZE, WE WOULD DO AND 20
AND 21, THAT'S WHEN WE WERE
AWARDED THE NIH SBIR FAST TRACK
AWARD SO PHASE 1 AND PHASE 2 AND
THEN THE STTR AWARD WITH HIGH
YU, WE GOT IT IN 2021.
SO AS AN ENTREPRENEUR, THERE'S
NEW TECHNOLOGIES AND SEEING
EVERYBODY HERE, IT DOES BRING
SOME OF THIS MEMORY BACK BUT
THEN IN THE BACK OF MY MIND I'VE
ALSO EXPERIENCED SOME REALLY
SOME REALLY SCARY FOULS AND
LONG, LONG TOWN HILL TRAVELS,
TOO, BUT IT'S NOT ALL BAD
STORIES HERE.
I WILL POINT OUT EXCITING
RESULTS AND AND REALLY OUR
BACKGROUND WITH THE TIPS, WE
COLLABORATE WITH A LOT OF THESE
PHARMA COMPANIES.
PUBLISHED WITH PFIZER, SYNOFI
DID A WEBINAR FOR US AND ALSO WE
HAD A PUBLICATION FOR US.
AND THE [INDISCERNIBLE] THERE
ARE 700 LAPS TAKEN--THEY USE
THIS PRODUCT FOR DRUG AND THIS
IS A SERIES OF ENZYMES WE'RE
PLACING THIS TRANSFER ACES, THE
ACTIVATING GROUPS ARE THE
TRANSFER ACE AND THE
BIOSYNTHETIC ENZYMES.
YOU'VE ALREADY HASHED ABOUT
THOSE ENZYMES THAT ARE INVOLVED
IN THAT IN MOST OF THE ENZYME
WILL BE MANUFACTURED BY IMCs
AND COMMERCIALIZED SO FOR MORE
INFORMATION GO SEE THE POSTER
THAT DR. JOHN MACARTHUR WILL
PRESENT, HE DID HIS POST DOC
WITH C.CHEN AS WELL.
AND THE OTHER SET OF ENZYMES
THAT WILL BE COMMERCIALIZED ARE
ABOUT 10 DIFFERENT OTHER ENZYMES
THAT HELPS MAKE THE GLYCANS
HERE.
AND THE KIND OF THE COMPARISON
OF THE ENZYME SCALE UP THAT
WE'VE DONE IS THAT TYPICALLY IN
THE LAB SCALE, YOU DO YOUR FLASK
CULTURES AND WE'VE MIGRATED IT
TO A FED BATCH FERMENT AITIONZ
TECHNIQUES WITH OUR OWN MEDIA IN
THE CELLS AND WE TYPICALLY GET A
DESCENT 5 TO HUNDRED FOLD
IMPROVEMENT IN THE YEAR AND WE
WOULD TYPICALLY GO IN THE
QUAINTITYIS OF PRODUCTION FOR
THE LOW SCALE, AND AND THEN WE
HAVE TO MAKE SURE THEY ARE
PRODUCED IN THE FLASKS AS WELL
AS TRANSFERRED TO THE FED BATCH
SCALES ARE EQUIVALENT AND SO THE
PURITY OF BOTH THE FLASK
CULTURES AND THE FERMENTATION AS
SHOWN IN LANE 1 OR 2 FOR 1 OF
THE SILL O TRANSFER ACES, YOU
CAN SEE THE FERMENTATION METHOD
INTRODUCE EXTRA BANDS, THE
PURITY IS NOT AS PURE IN THE
SMALL SCALE BUT FOR BOTH SOLID
TRANSFER ACES OF THE SECOND
SOLID TRANSFER ACE OF LINK 2, 3
AND 4, THEY WILL HAVE COMPARABLE
LEVELS AND THE PERCENT
CONVERSION OF THE TRANSFER ACE
AND THE TRANSFERRING TO CMP 5 AC
IS EQUIV LEAPT WHETHER IT'S IN
PLAOF THESH R FLASK CULTURE, AS
YOU SEE IN THE TEAL AND THEN FOR
RED, THE SECOND TILE OF TRONS
FER ACE IS PRETTY EQUIVALENT
ACTIVITY WHETHER YOU CULTURE IT
IN THE FERMENTER OR IN THE
FLASK.
AND THEN THE--ONCE YOU GET THE
ENZYMES WE DO HAVE TO MAKE SURE
IT DOES GO OUT TO THE COMMUNITY.
AND WE HAVE TO MAKE SURE THAT IT
WORKS IN NOT ONLY GRADUATE
STUDENTS, I THINK JUSTIN POINTED
IT OUT THAT YOU MIGHT BE
SPENDING A HUNDRED DOLLARS ON AN
INN ZYME BUT IF THEY SPILL SOME
OR ACCIDENTALLY FREEZE SOME OR
ACTUALLY COOK IT, YOU DON'T WANT
IT TO BE INACTIVE AND YOU SPENT
THAT MONEY AND TIME PREPARING
FOR THAT REAGENT.
SO WE ACTUALLY GO PRETTY
EXTENSIVE ROUNDS OF FORMULATION
TO INSURE WE STABILIZE THAT
ENZYME.
SO HAVE YOU AN EXAMPLE HERE
HERE, DIFFERENT CONSULTS,
DIFFERENT BUFFERS AND TITRATIONS
AND WHAT'S PLOTTED IN THE Y-AXIS
HERE IS ELEVATED TEMPERATURE OR
MELT TEMPERATURES YOU HAVE A
MORE TABLE ENZYME.
THERE'S 1 INTERESTING POINT HERE
WITH THE ORANGE DOT ON THE LEFT
CORNER IS THAT THAT 1 PARTICULAR
SALT WITH THAT PH IS PRETTY
STABLE COMPARED TO EVERY OTHER
SALT WHERE IT TRENDS IN A
CONTINUOUS MANNER TOWARDS A MORE
BASIC PH, RIGHT?
BUT IF YOU COMPARE THAT
PARTICULAR SALT WITH OTHER
BUFFERS, EVEN AT THE HIGHER PH,
THAT SALT BUFFER DOES PROVIDE
MORE STABILITY FOR THAT ENZYME.
AND THEN AS IT GOES THROUGH
ROUNDS 1, 2, 3, 4, 5, YOU DO
SHIFT THAT MELTING AS SHOWN HERE
THIS IS A SINGLE WAVE LENGTH
LIGHT SCATTERING AT 256 WHERE IT
MEASURES NANO PARTICLES SO AS
THE PROTEIN UNFOLDS THE
PARTICULARRATES COME TOGETHER
AND IT WILL SCATTER LIGHT AT 266
AND YOU MAY ACROSS THE
TEMPERATURE AS THAT LIGHT
SCATTER INCREASES, YOU WANT TO
SEE THIS SHIFT MORE TO OUR
HIGHER TEMPERATURES, HENS MORE
STABLE PRODUCT, SO THIS IS YOUR
SURFACE PLATS YOU WILL GO WITH
YOUR PH AND YOUR SALT
CONCENTRATIONS, SO WITH THIS
CONSTRUCT A LOWER BUFFER SALT
WITH A HIGHER PH PROVIDED HIGHER
MELT TEMP.
SO A LOT OF THESE FORMULATIONS
WILL GO THROUGH FOR PRACTICALLY
21 DIFFERENT ENSWROIMS TO
SABLIZE IT AND THEN WE WOULD GO
THROUGH A LONG-TERM STABILITY
TEST, AT THERE DIFFERENT
TEMPERATURES AT MINIMUM SO HERE
IS 1 OF THE ENZYME CONSTRUCTS AT
NEGATIVE 20 DEGROOSS, 4-DEGREES
AND ROOM TEMPERATURE,
20-25-DEGREES, AND THE
ACTIVITIES ARE MEASURED BY 4
DIFFERENT ANALYSTS, CHEM KISTS,
ACROSS A 24 WEEK PERIOD.
SO ONCE YOU HAVE THE FORM AWGZ
WE HAVE A COMPONENT AND THE
ENZYME BETWEEN ENZYME A AND B AS
ALSO TO BE WORKED OUT SO THAT
HAVE YOU A ROBUST RANGE OF
PERFORMANCE SO HERE WHAT I'M
SHOW SUGGEST ABOUT A HUNDRED
MICROGAMS PER MILL CONCENTRATION
OF ENZYME A AND FURTHER DILUTED
10 FOLD AND THE RATIO OF ENZYME
B THAT'S TITRATED TO ENZYME A,
AND DEPEBDING ON THE TITRATION
YOU CAN HAVE A PRETTY EFFICIENT
CONVERSION OF THE ACTIVATED
SUGAR OR CAN YOU HAVE A PRETTY
LOW CONVERSION BUT YOU CAN SEE
THE DILUTION OF--LASER--NO.
THE ENZYME B IS NOT AS HIGHLY
REQUIRED.
A SMALL QUANTITY OF ENZYME B IS
THE PRICE.
MULTIPLIER IS 1 TO 1 RATIO OR
HUNDRED FOLD DILUTION, HUNDRED
FOLD DILUTION OF ENZYME BR IS
SUFFICIENT TO DRIVE THAT
REACTION AND ENZYME A, CAN YOU
ACTUALLY ACHIEVE YOUR REACTION
WITH 10 MICROGRAMS INSTEAD OF A
HUNDRED MICROGRAMS.
SO ULTIMATE GOAL MRILY TO
SUMMARIZE THE GRAND SCALE TO
KILOGRAM SCALE IS THE SWEET SPOT
FOR THE SYNTHETIC APPROACHES OR
ENZYMATIC SYNTHETIC APPROACHES
AND THEN WE'RE ENSURING THAT THE
ENZYMES ARE STABLE IN MULTIPLE
HANDS IT EVEN IN PROFESSOR'S
HANDS SO I KNOW GRAD STUDENTS
THEY DO A LOST WORK BUT, AND
SIMPLE TO EASY TO USE MOTE COLS,
SO THAT YOU GET ROBUSTY
REACTIONS, AND THE OPME GETS AN
AFFORDABLE REACTION SO THAT YOU
CAN MAKE HUNDREDS OF MILLIGRAMS
POTENTIALLY GRAMS OF THE
ASILATED GLYCANS AND AND THEN OF
COURSE PROFESSOR CHEN HOUNDS ME
ON THIS 1 AND I ADHERE ON IT
BECAUSE I MYSELF AM AN
ATTESTAMENT TO THIS, I AM A
NONSPECIALIST PROMISING TO MAKE
GANGLIO CITES AND OLIGO
SACCHARIDES.
SO I WOULD LIKE TO ACKNOWLEDGE
THE NIH PROGRAM, THE STTR, THE
R42 AND THE R44.
THAT'S THE SBIR AWARD AND THEN
THE IMCS TEAM THAT'S PICTURED
HERE THAT'S WORKING ON THE
VARIOUS ENZYMES HERE AND THEN
THE UCDAVIS, I KNOW THIS IS A
LOT SHORTER BUT I DID NOT GET
THE FULL LIST OF NAMES AND I
KNOW THERE'S PLENTY OF MORE
THAT'S ASSOCIATED WITH OUR UC
DAVIS TEAM.
SO THANK YOU SO MUCH
[ APPLAUSE ]
>> THANK YOU ANDREW FOR
REMINDING US THAT WE'RE ALL CUTE
IN SCIENCE IN 1 WAY OR ANOTHER.
SO THE NEXT TALK IS FROM RON
SCHNAAR, JOHNS HOPKINS SCHOOL OF
MEDICINE, HE WILL TELL US ABOUT
GANGLIO SIDE INTERACT OHM TOOL
KIT.
>> I HAVE SOME THANK YOUS, I
WANT TO THANK THE ENTIRE GROUP
UP HERE AND DOUG AND AMANDATORY
AND PAM.
MODEL CITIZEN HAVE BEEN THE FACE
OF THIS CONFERENCE.
I WANT TO THANK CHEN FOR
INTRODUCING GANGLIOICIDES SO I
DON'T HAVE TO DO AS MUCH OF
THAT.
AND FINELE, I HATE BEING THE
SPEAKER BEFORE LUNCH SO I WANT
TO THANK UMESH DESAI WHO'S AFTER
ME, WHOOPS, BACK.
>> THIS IS OUR TEAM.
THEY ARE HERE AND WILL BE
MANNING POSTER NUMBER 7 FOR
DETAILS OF WHERE WE'VE GONE WITH
THIS, SO THERE'S NO
GANGLIOICIDES, SO WE'RE
INTERESTED IN THEIR FUNCTIONS.
SO OF COURSE THIS IS JAM 3,
YOU'VE SEEN IT ALREADY, IT AS
THRI KAN--KANAS ATTACHED TO A
CERAMIDE AS YOU HEARD,
IMPORTANTLY, OF THE MANY
GANGLIOICIDES THAT ARE
AVAILABLE, IN NATURE THESE 8 ON
THE BOTTOM, I'M NOT GOING TO USE
THAT, I DON'T SEE THE OTHER 1
HERE.
THESE 8 DRIVE A LOT OF THE
FUNCTION THAT WE KNOW OF.
THESE 4G M1, DT1 A AND B, ARE
THE MAJOR GLYCANS OF THE BRAIN.
GM3 ASK A NONNEURONAL
GANGLIOICIDE INVOLVED IN
CONTROLLING METABOLISM AND AS AN
EXAMPLE GD2 IS A THERAPEUTIC
CANCER TARGET WITH AN ANTIBODY
IN THE MARKET.
SO THERE'S MANY PLACES WHERE
THESE HAVE FUNCTIONS, AND
IMPORTANT TO UNDERSTANDING THOSE
FUNCTIONS IS UNDERSTANDING WHERE
THEY ARE, THEY'RE EMBEDDED DEEP
IN THE PLESES MA MEMBRANES
PRIMARILY HEADING OUT INTO THE
EXTRA CELLULAR SPACE.
THEY CAN BE RELATIVELY LARGE,
50-ANGSTROMS, OUT FROM THE
MEMBRANE WHERE THEY CAN'T HAVE
MULTIPLE FUNCTIONS AND THOSE
FUNCTIONS WE THINK OF IT BEING 2
TYPES.
FIRST OF ALL THIS IS A STAINING
OF WHAT HAPPENS TO NERVE CELLS
IN CULTURE FOR GANGLIO SEEDS GU1
A AND B, AND YOU CAN SEE IT'S
ALL OVER THEIR SURFACES AND ALL
OVER ALL OF THEIR MEMBRANE
EXTENSIONS.
AND IN THE MEMBRANE, THE GANGLIO
SIDS CAN ACT LAT ROOLLY WITH
MOLECULES IN THEIR OWN AND ON
THE MEMBRANE ON THE LEFT TO
MODIFY THE ACTIVITY OF THOSE
PROTEINS.
AND THIS HAPPENS FOR INSTANCE
FOR RECEPTOR TYROSEEN CAINS,IS
CONTROL THE INSULIN RECEPTOR OF
GROWTH REPRESENT SEPTORSOR TRACK
A AND FOR PERHAPS
NEUROTRANSMITTER RECEPTORS.
IN ADDITION, THEY CAN ACT AS
RECOGNITION COLKIEWLS FOR
PROTEINS THAT ARE IN THE EXTRA
CELLULAR SPACE OR OTHER WITH THE
AXON AND BACTERIAL TOXINS ARE
SOLUBLE PROTEINS THAT BIND TO
GANGLIOICIDES, SO THIS IS WHAT
OUR GOAL IS ABOUT, IT'S ABOUT
UNDERSTANDINGLET IRPT ACTION OF
GANGLIOICIDES WITH PROTEINS TO
DRIVE ACTIVITY, AND WE WANT TO
[INDISCERNIBLE] GANGLIOICIDE
INTERACT OHM, TO BE A BROADLY
APPLICABLE SET OF PHOTO AFFINITY
CLICK GANGLIOICIDES TO ADENTIFY
GANGLIO SIDE INTERACTING TEENS
IN VARIOUS, BIOLODGE 8 HOURSICAL
TON TEXTS.
THE TEAM HAVE SUCCESSED IN THESE
3 GOALS IF WE PUT THE TAG ON THE
FATTY ACID, PART OF THE SERIES
POINTS MID IN THE MEMBRANE.
AND THAT WE HAVE SHORT TAGS,
THAT HAVE A [INDISCERNIBLE] AND
AN L-CINE FOR PHOTO AFFINITY
LABELING AND CLICK CHEMISTRY,
THIS CAN BE ON THE GATE KEEPER
LACTOSE AS SHOWN, THE ACID, AND
JAM 1 TAG B, THERE'S THE LONG
CHAIN REPLACEMENT FOR THE FATTY
ACID.
YELL AT ME TIMEWISE, OKAY?
THIS IS JUST EXAMPLES, I WILL
NOT SHOW YOU ALL THE THINGS
WE'VE MADE BUT FOR EXAMPLE, JAM
1 UPPER READ ARROW ON THE LEFT
WE CAN TARGET THE SIDE CHAIN
WITH [INDISCERNIBLE].
TO GENERATE A UNIQUE ALDEHYDE
WHICH WE CAN CONVERT GOING DOWN
TO THE NEXT LINE TO THAT PRIMARY
AMINE AND THEN YOU JUST HAVE
PRIMARY AMINE TO INSTALL THE
L-KINE.
LIKEWISE, WEATHER ON THE TOP
LEFT, WE CAN TARGET THE GAL
ACTOSE 6 POSITION WITH THE GAL
ACTOSEOX DACE TO AGAIN MAKE A
UNIQUE ALDA HIDESSA HIDE,
CONVERTED TO AN AMINE AND
INSTALL OUR SHORT
[INDISCERNIBLE] THERE.
AND FINALLY, IN TERMS OF GENERAL
PROCEDURES, WE WERE LUCKY ENOUGH
TO GET AN EXPRESSING PLAZ MIDS
MIDOF SINGLE LIPID SERIES POINTS
MID MDA SOLACE WHICH CLIPS THE
FATTY ACID OFF, LEAVING US
PRIMARY AMINE AND 2 STEPS WE CAN
GO FROM THE LYSOGANG LIAISONEE
SID TO OUR IN TAG B.
ALL OF THESE HAVE BEEN PRODUCED
AND FINALLY WE CAN USE THE
SYNTHESIS, YOU HEARD SO MUCH
ABOUT TO MAKE MORE COMPLEX SCAN
OUT OF SIMPLER 1S.
SAMPLE HERE, GEM 1, TAG A, WE
TREAT WITH [INDISCERNIBLE],
TRANSFER ACE IN THE PRESENCE OF
SIALOGLYCANSIC ACID TO MAKE GD1
A [INDISCERNIBLE] TAG A.
AND OF COURSE WE DO THAT FOR TAG
B SO WE CAN EXPAND OUTWARDS IN
THIS WAY, USING CHEMO AND
SEMANTICS SYNTHESIS.
THESE ARE CHARACTERIZED BY THIN
LAYER CHROMATOGRAPHY ON THE
LEFT.
WE HAVE A NICE PURIFICATION
PROCEDURE THAT MITCHELL AND
GALLON HAVE OPTIMIZED PROTOON
AND CARBON NMR AND MASS
SPECTROMETRY, SO THESE ARE
CHARACTERIZED TAGS.
TO DELIVER THE CELL IS A TRICK,
BECAUSE IF YOU TAKE GANGLIO SIDS
THEY MAKE NICE CELLS AND IF YOU
ADD THEM, THEY GET TAKEN UP AND
SENT TO THE LYSOSOME, BUT WE HAD
FOUND IN OTHER STUDIES, THAT
DELIVERING THEM IN COMPLEX WITH
METHYL BATTA DEXTRAN SULFATE
TRON ON THE LEFT, DOES ENHANCE
THAT AND ENHANCES IT REALLY
WELL.
SO WE PREMIX THE METHYL BAIT
BATTA WITH THE PROBE, INCUBATE
IT WITH CELLS WASHED AND TO SEE
WHERE IT IS, WE CLICK THE ALKINE
WITH ALEKSA FLOOR
[INDISCERNIBLE].
AND THESE ARE THE BEAUTIFUL
IMAGES WITH THE CARCINOMA CELLS,
YOU CAN SEE IT LOOKS LIKEOOSE ON
THE SURFACE, ON THE RIGHT IS TAG
B DUG WAY DOWN INTO THE MEMBRANE
AT THE VERY END, IT DOES STAIN,
USING THIS PROCEDURE, BUT WE
KNOW IT'S THERE BECAUSE IT TAGS
PROTEINS.
GALLON AND MITCHELL WENT FURTHER
TO PROVE THIS IS ON THE SURFACE
BY TAKING GM 3 TAG A LOADED IN
THIS CASE, A431 SEMESTERS AND
TREATING THOSE WITH
SIALOGLYCANSITTICS WHICH DOESN'T
GET INSIDE THE CELLS AND THEY'RE
ALL ACCESSIBLE TO
[INDISCERNIBLE] AND THE TAG.
AND TO SHOW YOU THIS IS
TRANSFERAL BOTH TO OTHER CELL
TYPES, THIS IS NEUROBLASTOMA
CELLS, CAN YOU SEE THE DIFFERENT
MORPHOLOGY, BEAUTIFULLY STAINED
ON THE SURFACE.
STEP WOP SYNTHESIZE THEM, STEP
2, DELIVER THEM, STEP 3, TAG
PROTEINS SO WE'RE DOING, WE'RE
LOADING THE CELLS WITH OUR
METHYL CYCLE DEXTRIN GANG
BIOICIDE PROBE, WASHING THEM,
EXPOSING THEM TO UV TO ACTIVATE
THE PROTEINS, CLICK ON A BIOTIN
AICIDE SOLUBLIZE, ANALYZE AND
CAPTURE.
THIS IS AN EXAMPLE OF THE CELLS,
TREATED WITH THE 3 DIFFERENT
TAGS AT THE TOP FOR NO PROBE, WE
THEN SOLUBLIZE THE PROTEINS, RAN
THEM ON A GEL AND USED CHOLERA
TAXIN A WHICH RECOGNIZES JAM 1
AS THE PROBE AND CAN YOU SEE, WE
PICKED UP SOME NUMBER OF
PROTEINS IN THE HUNDRED TO 175
KILODALTON RANGE THAT
INTERACTING FROM THESE CELLS IF
WE--I SHOULD BACK AND SAY, YOU
SEE NO TRAINING WITH GM3 OR A 1
TAG A AND TAG B PICK UP
DIFFERENT PROTEINS FOR JAM 1
THOSE RIGHT DOWN AT THE BOTTOM.
WE BELIEVE WE HAVEN'T CONFIRMED
THIS, MAY BE SMALL TETRASPANINS,
THAT'S IN HOCKA MAURI OSHEROFFY
PROPOSED INTERACTING WITH
GAIPGLIOICIDES IN THE PLANE OF
THE IDEA TAG B AND TRICK THE
[INDISCERNIBLE] AND THEY ARE NOW
AT THE POINT OF CAPTURING FOR
THE [INDISCERNIBLE].
WITH THAT I WILL FINISH, PLEASE
GO TO POSTER 7, THIS WORK HAS
REALLY BEEN DRIVEN BY 2 PEOPLE,
[INDISCERNIBLE] AND MITCHELL
PORTER, AND WE HAVE HAD
ESSENTIAL TOOLS PROVIDED TO US
BY DR. CHEN AS MENTIONED AND
ANDROGEN LIEU LEE WHO WE JUST
HEARD FROM AND MACARTHUR AT.
>> AND NOW FOR THE LAST SPEAKER
BEFORE LUNCH--
>> WE HAVE 4 MORE MINUTES.
>> THE LAST SPEAKER IS UMESH
DESAI, WHO WILL TALK ABOUT TOOL
FOR PREDICTING GRIEK O
SOPHISTICATED MINE DPLI
KAN--KANA RECOGNITION OF
PROTEINS.
>> HELLO EVERYBODY, I WOULD LIKE
TO BEGIN BY THANKING PAM AND THE
CALL AND AMANDA AND EVERYBODY
CONNECTED WITH THE COMMON FUND
TO GIVE ME THIS OPPORTUNITY TO
ESSENTIALLY TELL YOU ABOUT WHAT
WE ACCOMPLISHED OVER THE LAST 3
YEARS AND ALSO IBDICATE TO WHERE
WE ARE HEADING SO, THE
SPECIFICALLY TRYING TO MOVE THE
IDEA THAT GLYCO GLYCANS CAN
ACTUALLY SERVE WELL AS DRUGS
BECAUSE THERE ARE VERY, VERY FEW
THAT ARE YOU KNOW HAVE SUCCEEDED
AS DRUGS AND I THINK WE THESE
WILL MAKE IT MORE FEASIBLE AND
THE SECOND PART OF THIS PROGRAM
WAS TO USE THIS TOOL AND
ESSENTIALLY HELP YOU KNOW
BIOLOGISTS WHO ARE NOT REALLY
TOO FAMILIAR WITH THE
COMPUTATIONAL ASPECTS TO COME UP
WITH IN BIOLOGY, SO THIS IS
ACTUALLY A 20 STEP TO BIG FOR
MOST PEOPLE BUT IF YOU REALLY
THINK FROM A BIOLOGIST
PERSPECTIVE THAT WHAT A
BIOLOGIST WANT TO KNOW HAVING
NOT SO MUCH UNDERSTANDING ABOUT
YOU KNOW GAGS AND THINGS OF THAT
SORT, NUMBER 1, YOU KNOW DOES MY
PARTICULAR PROTEIN BIND TO GAGS,
THAT'S THE BASIC QUESTION, IF IT
DOES BIND TO GAG THEN WHERE DOES
IT BIND, IS THERE A SPECIFIC
SORT OF SIGHT OF BINDING THEN
WHAT GAG WOULD PREFERENTIALLY
BIND TO THAT PARTICULAR SITE?
AND ASSUMING THAT ALL OF THESE
ANSWERS COME THROUGH, TRYING TO
UNDERSTAND AS TO WHETHER INVIVO
IN REAL LIFE SITUATION WHERE
THAT COMPLEX REMAINS STABLE AND
IS LIKELY TO INDUCE THE FUNCTION
THAT WE THINK IS LIKELY TO
INDUCE, SO THE WE HAVE
DEVELOPED--WHOOPS LET'S GO BACK.
TRIES TO ADDRESS THE 3 QUESTIONS
THAT ARE HIGHLIGHTED HERE, DOES
MY PROTEIN BIND TO GAG, IS A GAG
SITE IS WHAT WOULD
PREFERENTIALLY RECOGNIZE THAT IS
IT'S PULT MULTIPLE STEPS BUT WE
HAVE TRIED TO AUTOMATE IT IN
SUCH A FASHION THAT WE CAN
AUTOMATE IT IN A LIBRARY OF
SEQUENCES SOMEWHERE AROUND A
THOUSAND THOUSAND DIFFERENT
SEQUENCES.
SO THIS IS ESSENTIALLY THE
BOTTOM LINE IN TERMS OF WHAT THE
ALGORITHM TRIES TO DO.
NUMBER 1 IT BUILDS A LIBRARY OF
SEQUENCES THAT YOU WANT--AND
THESE SEQUENCES ARE AVAILABLE
FROM [INDISCERNIBLE] AND DPLI
KAN--KANA THERAPEUTICS AND WE
HOPE WE CAN ADD ANYBODY WHO
MAKES THESE GAG SEQUENCES
COMMERCIALLY AVAILABLE, WE WOULD
ADD IN THE FUTURE.
WE WOULD SCREEN ASSUMING WE
DON'T KNOW ANYTHING ABOUT GAG
INTERACTING WITH THE PROTEIN,
ANYTHING ABOUT PROTEIN BINDING
SITES WE VALID TO SCREEN THE
ENTIRE PROTEIN SURFACE FOR THE
POSSIBILITY OF GAG BINDING SO
DO--MULTIPLE BINDING SIGHTS AND
THEN OBVIOUSLY DO A WHOLE LOT OF
COMPUTATIONAL WORK, WE HAVE
DEVELOPED A TECHNOLOGY OF USING
2 DIFFERENT FILTERS AND
TRADITIONALLY COMPUATIONAL WORK
UTILIZES 1 FILTERS, WE HAVE DONE
2 FILTERS BECAUSE OF THE NUANCES
ABOUT GAG PROTEIN INTERACTS
OF--WHICH MIGHT EXIST INTO
NATURE IN 2 BINS, 1 IS A BIN
WHERE YOU HAVE SPECIFIC
SEQUENCES, THE SECOND WOULD BE
NONSPECIFIC SEQUENCES, JUST
BECAUSE I AM A MEDICINAL
CHEMIST, I'M A DRUG DISCOVERY
PERSON, I'M MORE ABOUTED IN
THESE SEQUENCES BECAUSE THERE IS
A TENDENCY TO CONVERT THEM INTO
POSSIBILITIES PRETTY HIGH TO
CONSECRETER THEM INTO DRUGS.
UNDER ANY CIRCUMSTANCES THIS IS
THE TOOL THAT IS AVAILABLE FOR
ALL NONPROFIT/EDUCATIONAL
INSTITUTION--AND THIS OPERATION
OF THIS WEBSITE ACTUALLY
PRESENTED IN THE PAST SO I'M NOT
GOING TO GO INTO DETAIL BUT THE
BOTTOM LINE IS THAT THERE IS A
TUTORIAL THAT IS AVAILABLE,
THAT'S THE TUTORIAL, YOU LOG ON
TO THAT SITE, YOU CREATE A USER
NAME, YOU USE THE TUTORIAL, TO
ESSENTIALLY YOU KNOW SET UP THE
INDIVIDUAL RUNS AND IT'S
ESSENTIALLY FULLY AUTOMATED,
THERE ARE ONLY 5 STEPS, THE KEY
ASPECT IS REALLY INTERPRETATION
AND THAT'S WHERE YOU PROBABLY
NEED TO READ SOME OF THE PAPERS
THAT WE'RE PUBLISHED ON THE
TOPIC OF HOW TO REALLY
INTEGRATE.
BUT, AS OF NOW, I THINK WE PUT
THIS UP IN A YEAR, MAYBE 14
MONTHS AGO AND WE HAVE HAD 28
UNIQUE USERS, ABOUT 283 JOBS AS
OF LAST WEEK, YOU KNOW AND I
THINK I AM EXTREMELY HAPPY THAT
AN UNDERGRADUATE STUDENT WAS
ESSENTIALLY ABLE TO DO THE
RESEARCH USING THE WEB TOOL WITH
PRACTICALLY NO, YOU KNOW INPUT,
PRACTICALLY NO INPUT THAT
DOESN'T MEAN NO INPUT BUT
PRACTICALLY NO INPUT FROM 1 OF
MY LEADING SCIENTISTS.
FOURTEEN INSTITUTIONS IN THE
U.S. HAVE USED IT, THESE ARE
ACROSS THE ENTIRE U.S., FOR
VARIOUS PURPOSES WE DON'T KNOW
EXACTLY WHAT EXPERIMENTS THEY
WERE DOG BUT HOPE LOOFULLY THEY
GOT SOMETHING GOT OUT OF IT AND
WE WILL SEE THAT THIS PARTICULAR
WEB SERVER WILL BE RECOGNIZED IN
FUTURE PUBLICATIONS.
AS FAR AS MY GROUP AND
ESSENTIALLY TRYING TO HELP
BIOLOGISTS WE HAVE STUDIED
SEVERAL DIFFERENT PROTEINS, YOU
KNOW STUDIED A BUNCH OF
PROTEINS, 1 OF THE LAST, 2
YEARS, 1 AND HALF YEARS, WE HAVE
DONE YOU KNOW WORK ON THE LAST
FEW DAYS, TRANSFER ACE 1, CHEMO
KINES AND YOU HEARD TALK FROM
[INDISCERNIBLE] YESTERDAY ABOUT
SOME OF THE WORK THAT WE HAVE
DONE WITH HIM.
TGF BETA 2, SPIKE GLYCO PROTEIN
FOR SARS-COV-2, WE HAVE A PAPER
IN CURRENTLY IN REVIEW WITH
IGF 1 R, WITH IS A TYROSINE
KINASE, THAT BINDS TO IGF1 R AND
NRP 1 WHICH IS CURRENTLY IN
PROGRESS.
THESE ARE THE
BIOLOGISTS/BIOCHEMISTS THAT WE
HAVE TRIED TO HELP, SHE WORKS ON
CYSTIC FIBROSIS AND SOME OF YOU
MAY KNOW HIM WITH REGARD TO THE
TRANSFER ACE, KRISHNA, YOU HEARD
HIM, AND [INDISCERNIBLE] WORKS
ON SARS-COV-2, P A TEL IS
WORKING ON IGF-1 R AND
[INDISCERNIBLE].
ESSENTIALLY WHAT DOES THE TOOL
TRY TO HELP?
HAD AND THIS IS PRIMARILY WITH
BIOLOGIST IN PERSPECTIVE MARKED
FOR IDENTIFICATION LIFE THAT'S
WHAT THE TOOL TRIES TO HELP YOU,
TOO.
NUMBER BE IN 2 IT CAN OOH
DEBTIFY THE SITE OF BINDING
WHICH MEANS, THESE ARE A
CRITICAL BINDING AND WHAT SHOULD
A BIOLOGIST MUTATE IN ORDER TO
GO FURTHER DOWN THE LINE, IT CAN
PREDICT THE STRENGTH OF
INTERACTION WHICH IS THE
RELATIVE AFFINITY FOR MOST
PURPOSES AND THEN, YOU KNOW WHAT
MED A POTENTIAL MECHANISM BE,
WOULD IT BE AN ALOE STATIC
MECHANISM, WHAT ARE THE ROLE OF
THE LENGTH OF THE CHAIN, THINGS
OF THAT SORT SO ALL OF DWHEES
CAN BE ANSWERED USING THE TOOL
THAT YOU DEVELOP.
NOW I WANT TO HOPEFULLY THE 5
MINUTES I HAVE, I WILL TELL YOU
ABOUT A VERY INTERESTING STORY
PRIMARILY BECAUSE IT ALERTS
BIOLOGIST AS WELL AS CHEMISTS TO
NOT ASSUME ANY [INDISCERNIBLE].
AND WHICH WAS DONE WITH
PHYSICIAN SCIENTISTS, AND THESE
KINASES AND YOU KNOW THEY DO
THIS BY VARIOUS MECHANISMS,
DIRECT BINDING TO THE RTK, OR
BINDING TO THE LIGAND AND THEN
MODULATING THINGS OF THAT SORT
AND VARIOUS, YOU KNOW RDKs
HAVE BEEN, YOU KNOW STUDIED FOR
THIS FDFR, AND ABOUT GAG BINDING
TO IGFR, AND IGF 1 SYSTEM AND WE
KNOW IGF1 R AND 1 IS IMPORTANT
BOTHOT CANCER PERSPECTIVE AS
WELL AS FROM GNLT GROWTH
PERSPECTIVE AND AGING
PERSPECTACLIVE, THINGS OF THAT
SORT, THE UNIQUE THING ABOUT IGF
1 R IS IT'S A CONSTITTATIVE
DIMER AND THE WAY IN WHICH
ACTIVATION WORK SYSTEM THAT THE
TRANSMEMBRANE HAVE TO CROSS EACH
OTHER, BRING THE KINASE DOMAINS
IN THE CELL CLOTHE TOGETHER IN
ORDER TO ENGAGE
AUTOPHOSPHORYLATION.
NOW, I GF1, 1 MOLECULE OF IGF 1,
1 IGFR TO 1 IGF1 IS A DIMER SO
MONITOR OR
MERIGFWOBDERFUL--WONDERFUL,
WOULD ACTUALLY DO YOU KNOW
ACTIVATION.
WHAT OF THE ROLE OF HS,
COMPLETELY UNKNOWN BECAUSE OF
OUR PRIOR WORK WITH DR. PATHWAY
GIVES TELETHAT WE HAD KNOWN THAT
HS WOULD ENGAGE IN, WOULD DO
SOMETHING, WE ARE NOT REALLY
UNDERSTOOD IT FULLY.
SO WE ASKED A QUESTION WHETHER
OLIGIO SACCHARIDES CAN DO SOME
TRICK AND WHAT IS THE CHAIN
DEPENDENCE AND WE HAD
INVESTIGATED THIS, WE HAD
PUBLISHED THIS SOMETIME AGO, SO
I'M SIMPLY PRESENTING THE
COMPUTATIONAL ASPECT BECAUSE YOU
WANT TO SEE HOW THIS HAS HELPED
THE BIOLOGIST OUT BUT BASICALLY
IGF 1 R, THE ACTIVE DOMAIN IS
EXTENSIVE, CONSISTS OF L1
DOMAIN, CRDOMAIN AND L2 DOMAIN
AND ESSENTIA WILY NOTHING WAS
KNOWN ABOUT GAG BINDING SO WE
DECIDED THE ENTIRE PROTEIN INTO
THE BINDING SITES.
BS1 TO-7 AND ESSENTIALLY
DR. [INDISCERNIBLE] OF
DISCIPLINARY SACCHARIDES,
HEXASACCHARIDES, OCT O
SACCHARIDES ON THESE ARE SITES,
USING THE 2 FILTERS, AND AND TO
PUT THE WHOLE SORT OF STORY, THE
BOTTOM LINE OF THE STORY
INTEGRAL EXPERIMENTS OVER A
FAIRLY LONG PERIOD OF TIME AND
THIS IS THE UNUSUAL PART THAT WE
FOUND THAT IS THE PREDICTION
WISE THAT THE HEXASACCHARIDE
BINDS IN AN INTERFACE BETWEEN
BS2 SQUARELY FIT BS 7 IN REAL
LIFE ISHT PHASE BETWEEN THE L1
DOMAIN AND THE CR DOMAIN.
QUITE THE OPPOSITE, AND WHAT IT
ACTUALLY MEANT WAS THAT LONGER
OLIGIO SACCHARIDES DO NOT BIND
IN THIS PARTICULAR SITE.
THEY ACTUALLY PREFER TO BIND IN
A SITE COMPLETELY DIFFERENT FROM
THE HEXASACCHARIDE.
NOW THIS IS THE REVEALING
PORTION.
WHY?
BECAUSE GENERALLY SPEAKING WE
CHEMISTS AS WELL AS BIOLOGISTS
ASSUME THAT A SACCHARIDES BINDS
IN A PARTICULAR SITE, IT WILL
BIND WITH THE SAME SITE, THE
HEXASACCHARIDE BINDS WITH THE
HIGH AFUNNITY OF THE SAME SITE,
THAT IS NOT REALLY TRUE AS FAR
AS PREDICTION GOES, WITH CVLS,
THIS ALSO FITTED WELL WITH THE
BIOLOGY DATA WE HAD WHICH YOU
KNOW WE HAD PUBLISHED NOW, IN
TERMS OF THE STRENGTHS OF
TECHNIQUE, THE TECHNIQUE COULD
NOT ONLY PREDICT THE SITE OF
BINDING BUT IT COULD IDENTIFY
RESIDUES THAT ARE IMPORTANT FOR
BINDING THAT 1 CAN ACTUALLY
MUTATE AS TO WHAT WOULD THEIR
CONTRIBUTION BE, SO BRINGING THE
WHOLE SORT EVER STORY SHORT AND
I'M PRESENTING ONLY VERY SMALL
PIECE OF DATA TO SUPPORT THE
CDLS PREDICTION, THAT WE STARTED
YOU KNOW A BUNCH OF,
THERMODYNAMIC AFFINITY STUDIESS,
USING FLUORESCENCE AND USING
TECHNIQUES AND THE BOTTOM LINE
IS AS YOU CAN SEE IN THE PANEL
C, SO THE PANEL C HAS 2 LINES, 1
IS RED, AND 1 ISIBLY, 1 STANDS
HEAR HSO6, WHEN IS THE
HEXASACCHARIDE, THE HS12 IS THE
[INDISCERNIBLE] SACCHARIDE AND
YOU CAN SEE THAT THE AFFINITY
INCREASES FOR THE HEXASACCHARIDE
AS THE CONCENTRATION IGF1
INCREASES AND THIS IS TYPICALLY
TRUE FOR COMPETITION.
FOR COMPETITIVE BIND
THANKSGIVING IS REALLY TRUE AND
FOR HS12, ESSENTIALLY THIS IS NO
COMPETITION, THE AFFINITY DOES
NOT CHANGE AT ALL.
WHICH MEANS THAT THEA SITE OF
BINDING ARE COMPLETELY
DIFFERENT, ALTHOUGH WE ARE ONLY
TALKING ABOUT THE ECTODOMAIN, SO
THIS IS THE REVEALING PART, IT'S
ACTUALLY A PRINCIPLE THAT I
THINK WE ALL TO BE FAMILIAR
WITH, NOT REALLY JUSTA ASSUME
THAT THE OLIGO SACCHARIDES BIND
IN A REPET TEF MANNER AND
STATISTICAL INCREASE AND
AFFINITY ARISES FROM IN FACT IN
THIS PARTICULAR CASE, HS 12
BINDS WITH THE WEAKER AFFINITY
UNDER CERTAIN CONDITIONS THAN
HSO 6, HSO 6 BINDS TIGHTER THAN
12, THE LONGER SACCHARIDE BINDS
WEAKER AND THE SHORTER 1 BINDS.
NOW BRINGS THE STORY TO A CLOSE
AT THIS POINT, YOU KNOW WE
MUTATED THE 2 RESIDUES THAT WE
TALKED ABOUT VERY IMPORTANT
WHICH IS R-GENINE 1 AND 38, AND
WE FOUND A SIGNIFICANT INCREASE
IN THE AFFINITY FOR THE
HEXASACCHARIDE.
THIS MEANS THE HYPOTHESIS, THE
PREDICTION APPEARS TO BE
REASONABLY CORRECT, WHAT I'M NOT
SHOWING YOU IS ALL THE IMMUNO
FLUORESCENCE DATA THAT SUPPORT
THIS IS IDEA AND THE PAPER IS
CURRENTLY IN REVIEW.
ANYWAY, I THINK OVER ALL WHAT I
WOULD LIKE TO SAY IS THAT THIS
U1 AWARD HAS BEEN VERY MUCH
BENEFICIAL TO US, WE WOULD HAVE
NEVER EMBARK OFFICE OF DIVERSITY
DEVELOPING A WEB SERVER AND I
SAID THIS EARLIER, TOO, BECAUSE
IT IS SOMETHING THAT YOU KNOW WE
WOULD NOT REALLY THINK ABOUT,
BUT THE UO1 AWARD MADE IT
POSSIBLE BY HIDING APPROPRIATE
WEB DEVELOPERS, AND THINGS OF
THAT SORT.
THE SITE ADDRESSES AS SHOWN
HERE, WE HAVE STUDIED VARIOUS
SYSTEMS, I WOULD SAY WE HAVE NOW
STARTED PROBABLY DOZEN AND HALF
PROTEINS AND THE ALGORITHM WORKS
REALLY WELL ACROSS VERY, VERY
DIFFERENT TYPES OF PROTEINS AND
RECEPTORS, AND YOU KNOW SO MANY
OTHERS AND WE THIS WILL CONTINUE
FORWARD.
WE WOULD REALLY WELCOME OTHER
GRUPS WHO ASSESS THE ROLE OF
GAGS IN THEIR SYSTEM AS WELL AS
YOU KNOW IF THERE IS ANY
COLLABORATIVE EFFORT THAT THEY
WOULD LIKE TO ENGAGE IN, WE WILL
WELCOME THAT, TOO.
FINALLY, THIS IS MY GROUP.
IT'S A FAIRLY OLD FORMULARY
REFERENCE FILE BUT I WOULD LIKE
TO HIGHLIGHT THE SPECIAL ROLE OF
2 INDIVIDUALS WHO DEVELOPED THE
WEB SERVER AND HELPED THE
BIOLOGIST, THE PERSON IN RED
T-SHIRT ON RIGHT IS
[INDISCERNIBLE] AS WELL AS THE
YELLOW T-SHIRT, ARE THE TOO
COMPUTATIONAL BIOLOGIST WHO DID
A LOT OF WORK.
THANKS A LOT.
I KNOW I AM PREVEBTING YOU FROM
HAVING GOOD LUNCH BUT HOPEFULLY
WITH THIS YOU KNOW YOU HAVE
ENJOYED MY TALK AND GREATLY
APPRECIATE IT.
[ APPLAUSE ]
LAST STEP, THE ENZYME THAT
CATALYZING THE LAST STEP IN THE
PATHWAY, IN ADDITION TO
CHANGESES IN ABUNDANCE WE ALSO
SEE INCREASED ABUNDANCE OF THE
OVAL CONNECT TRANSFERASE.
SPEAKING OF THE ROLE OF
O-GlcNac IN POTENTIATING
TUMORIGENESIS, IN ADDITION TO
GFAT WITH FIVE DAY ABBREVIATED
TO DON REDUCES TUMOR LOAD AS
SHOWN IN THIS UPPER RIGHT HAND
PANEL. THESE DATA ARE
REINFORCED BY GENETIC STUDIES
UTILIZING A KNOCK OUT OF THE
O-GlcNac TRANSFERASE IN
LUNG TISSUE SHOWN IN THE GREEN,
WHICH ENHANCES TUMOR FREE
SURVIVAL WHEN COMPARED TO MOCK
TREATED ANIMAL SHOWN IN THE BLUE
BY ANIMALS TREATED WITH AN
INHIBITOR THIGH MATE G WHICH
ENHANCES O-GlcNac LEVELS
BY INHIBITING THE O-GlcNac
AIS. THESE MODULATING
O-GlcNac CELL CULTURE IN
VIVO IS NOT WITHOUT CHALLENGE.
INHIBITORS OF THE O-GlcNac
HAVE BEEN TREMENDOUSLY POWERFUL
AS HAVE MURINE MODELS THAT
ENABLE TO DELETE OGT AND OGA
FROM SPECIFIC CELLS. HOWEVER,
OFTEN THESE APPROACHES LACK
TISSUE SPECIFICITY AND CAN BE
OFF TARGET EFFECTS AND THIS IS
ESPECIALLY TRUE OF CURRENT HBPN
INHIBITORS. OVEREXPRESSING OGT
AND OGA AS WELL AS DELETING THEM
CAN BE LETHAL LETHAL. AND AS JEN AND
COLLABORATORS ARE EXPLOITING,
EYE BUN DANCE OF ENZYMES IS
ITSELF REGULATED BY
O-GlcNac LEVELS. THE
LATTER IS CRITICAL IN ADDITION
TO CATALYTIC ACTIVITY BOTH
ENZYMES ARE BELIEVED TO PLAY
SCAFFOLDING ROLES IN THE CELL.
THUS THE GOALS OF OUTCOME OUR
COMMON FUND SUPPORT WERE TO
GENERATE GENETICALLY ENCODED OGT
AND OGA CONSTRUCTS THAT ALLOW
SMALL MOLECULE REGULATION OF
O-GlcNac LEVELS. THE
APPROACH WE CHOSE RELIES ON
DESTABILIZATION OF THE MAIN
TECHNOLOGY DEVELOPED IN THE LAB
AT STANFORD. HERE FUSION OF
DESTABILIZATION DOMAIN BASED ON
FKB 12 OR HYDRO FOLATE REDUCTASE
TO PROTEIN OF INTEREST RESULTS
IN FUSION PROTEIN DEGRADATION.
THESE DESTABILIZATION DOMAINS
ARE MUTATED SUCH THAT BINDING TO
LIGAND EITHER SHIELD OR TRIMETH
PRONE STABILIZES THE
DESTABILIZATION DOMAIN AND IN
TURN THAT OF FUSION PROTEIN.
THIS APPROACH ALLOWS RAPID DOSE
DEPENDENT INDUCTION OF PROTEIN
-- INDUCTION OF PROTEIN
ABUNDANCE INDEPENDENT OPHIURANS
SUBSCRIPTIONAL AND
POSTTRANSCRIPTIONAL REGULATION.
AS TOOLS ARE GENETIC IT IS
POSSIBLE TO GENERATE INDUCIBLE
SYSTEMS ALLEVIATING OFF TARGET
EFFECTS OF VIRAL TRANSDUCTION,
TRANSIENT TRAN FECKS AND SHORT
HAIR PIN SHORT INTERFERON RNA
KNOCK DOWN. IT IS ALSO POSSIBLE
TO WASH THE DRUGS OUT PROVIDING
CRITICAL CONTROLS. EXAMPLE OF
DESTABILIZED OGA IS SHOWN HERE
IN LOWER PANEL DEMONSTRATING
PROBUST CHANGES IN OGA ABUNDANCE
IN ONLY FOUR HOURS AS WELL AS
CHANGES IN THE ACTIVITY OF
TESTIFY O-GlcNac AIS WITH
PROOF OF PRINCIPAL IN HAND WE
DEVELOPED A SUITE OF TUMORS TO
ALLOW RESEARCHERS TO ADDRESS A
BROAD RANGE OF BIOLOGICAL
QUESTIONS. O-GlcNacIST
OUR OVERALL STRATEGY IS DEPICTED
ON THIS SLIDE. OUR STRATEGY
RELIES ON USING THE GATEWAY
RECOMBINATION SYSTEM. WE HAVE
GENERATE EIGHT DESTINATION
VECTORS WHICH EPICODE TUNING AS
WELL AS -- ENCODE TUNEK AS WELL
AS VECTORS WHICH INCLUDE OGA OGT
AND ENZYME WITNESS THE HBP. USE
OF REBOMB BY NATION GENERATES --
RECOMBINATION GENERATES OUR
CODING SEQUENCE INTO OUR
DESTINATION VECTOR. THESE ARE
ALL THEN SEQUENCED AND THE
ABILITY OF CONSTRUCTS TO BE
STABILIZED BY TRIMETHPRONAL
SHIELD AND IMPACT O-GlcNac
LEVELS IS 964 PLATE FORMAT.
SUBSEQUENTLY WE SELECT THE MOST
TRACTABLE CONSTRUCTS MAKING
STABLE CELLS AND THESE ARE THEN
CHARACTERIZED USING MORE
CLASSICAL APPROACHES. OVER THE
NEXT FEW SLIDES I WANT TO
HIGHLIGHT JUST A FEW KEY POINTS
ASSOCIATED WITH EACH STEP IN
THIS PROCESS. FIRST WE
DEVELOPED EIGHT DESTINATION
VECTORS BASED ON TWO
COMMERCIALLY AVAILABLE
DESTINATION VECTORS, THAT ENCODE
RECOMBINATION CASSETTE SO WHERE
OUR CODING SEQUENCE IS
INTRODUCED FUSED EITHER TO GFP
-- FUSED TO GFP AND V 5 EPITOPE.
SO IN B YOU CAN SEE THAT WE HAVE
INTRODUCED EITHER DI HYDRO
FOLATE REDUCTASE OR FKBP INSTEAD
OF GFP ENABLING SIMPLE TUNING.
IN C WE HAVE INTRODUCED DIHYDRO
FOLATE AND FKBP FIVE PRIME OR
THREE PRIME TO GFP, ALLOWING
BOTH TUNING OF THIS CONSTRUCT
BUT ALSO CRACKING OF THE
CONSTRUCT IN VIVO. FINALLY IN D,
WE HAVE INTRODUCED THE DI HYDRO
FOLATE REDUCTASE IN FKBP
DOWNSTREAM OF 2A SEQUENCE WHICH
IS THREE PRIMED TO GFP. THESE
CONSTRUCTS ARE ANTICIPATED TO
ALLOW TUNING WITHOUT GENERATING
A LONG FUSION PROTEIN. HOWEVER,
THE GFP CAN BE USED TO CONFIRM
TRANSFECTION EFFICIENCY.
COMPLIMENTING DESTINATION
VECTORS ARE 40 ENTRY VECTORS
ENCODING THE MAJOR VARIANTS OF
OGT OGA UAP AND G FAT. ALLOWING
RESEARCHERS TO PROBE THE ROLES
AND REGULATION OF EACH PROTEIN
VARIANT. IN ADDITION TO THE
DIFFERENT ISOFORMS WE HAVE ALSO
GENERATED CATALYTICALLY
INEFFICIENT OR DEAD CONSTRUCTS.
AS WELL AS MUTATIONS
CHARACTERIZED BY THE COLO LAB
ANTICIPATED TO INCREASE
UTILIZATION OF UNNATURAL
SUBSTRATES. FINALLY WE HAVE
GENERATED A MUTATION AT GFAT
WHICH THERE IS NO FEEDBACK
INHIBITION BY UDP GLCNAC. THESE
ARE COMBINED TO GENERATE 400
GLYCOTUNERS SEQUENCE VERIFIED.
AND THEIR ABILITY TO BE
STABILIZED BY SHIELD AND TRIMETH
PRONE HAVE BEEN CHARACTERIZED IN
96 WELL PLATE FORMAT. IN SUM WE
DEVELOPED A SUITE OF TOOLS THAT
HAVE NUMEROUS APPLICATIONS, WE
HAVE GENERATED EIGHT TUNEABLE
DESTINATION VECTORS, 40 GATEWAY
COMPLIANT OG,T OGA GFAT AND UAP
CONSTRUCTS AS WELL AS RANGE OF
USEFUL MUTATIONS. THESE HAVE
BEEN COMBINED TO GENERATE 400
GLYCOTUNERS WHICH ARE MOST OF
THE WAY TO CHARACTERIZATION AND
GENERATED A RANGE OF VALIDATED
PROTOCOLS FOR DETECTION OF
O-GlcNac IN 96 WELL PLACE,
LIKELY GO WE ANTICIPATE THESE
TOOLS CAN BE COMBINED IN
DIFFERENT WAYS TO ANSWER
DIFFERENT BIOLOGICAL QUESTIONS.
WE THINK TUNING OF
O-GlcNac LEVELS IN CELL
CULTURE AND XENOGRAPH MODELS
WILL ENABLE RESEARCHERS TO
RECAPITULATE DISEASE MODELS. WE
ALSO THINK THAT THE MUTATIONS
THAT HAVE BUMPED OUT ACTIVE SIDE
WILL ALLOW THE OR IMPROVE THE
INCORPORATION OF UNNATURAL
SUGARS, FACILITATING THE
IDENTIFICATION OF GLYCOSYLATED
PROTEINS BY CHEMISTRY, OR
STUDYING THE DYNAMICS OF
GLYCOSYLATION. PROTEINS FUSED TO
GFP WE ANTICIPATE CAN BE TRAPPED
IN SPECIFIC SUB CELLULAR LOCI.
USING THE GFP WE THINK THAT THE
DIFFERENT ISOFORMS WILL ALLOW US
TO ASSESS DIFFERENT ISOFORM
FUNCTION AS WELL AS DIFFERENT
POST TRANSLATIONAL MODIFICATION.
THE TUNING VECTORS THAT WE HAVE
DEVELOPED CAN BE USED FOR
ANYTHING IN WHICH THIS APPROACH
WORKS SO TYPICALLY MOST PROTEINS
IN THE NUCLEUS AND CYTOPLASM.
WITH MY LAST FEW MINUTES I WANT
TO TELL YOU ABOUT HOW WE HAVE
BEEN APPLYING THESE VECTORS TO
STUDY THE IMPACT OF
O-GlcNac ON AUTOPHAGY.
THAT IS A HOMEOSTATIC PROCESS
THAT PROMOTES CELL SURVIVAL BY
REMOVING PROTEIN AGGREGATES AND
DAMAGED ORGANELLES. WE HAVE
BEEN INTERESTED IN ADDRESSING
THE HYPOTHESIS THAT
O-GlcNac MAY POTENTIATE
AUTOPHAGY, ESPECIALLY IN THE
HEART REDUCING CELL AND TISSUE
DEATH AT TIMES OF STRESS.
HOWEVER, ONE CHALLENGE THAT WE
FACE IS THAT ALL OF THE
APPROACHES THAT WE HAVE USED TO
MODULATE O-GlcNac LEVELS
GENETICALLY BY SI RNA AND VIRAL
TRANSDUCTION, HAVE ACTIVATED
AUTOPHAGY IN THE CONTROLS. SO
THIS WAS AN AWESOME PROJECT TO
TEST OUT OUR DIFFERENT TUNEABLE
CONSTRUCTS ON. SO THIS PROJECT
HAS BEEN LED BY CAMEL FOY A
RESEARCH ASSOCIATE IN THE LAB
AND WHAT CANAL HAS DONE IS
GENERATE H 9C 2 CELLS MYOBLAST
HEART LAKE CELLS THAT ARE STABLY
TRANSFECTED WITH YELLOW
FLUORESCENT PROTEIN OGT OR
CATALYTICALLY INEFFICIENT OGT IF
WE FOCUS ON THIS PANEL LABELED
LC 3 WE ARE ASSESSING CONVERSION
OF LC 3 FROM ITS UNLIP DATED
FORM TO LIP DATED FORM LC 32 AS
MEASURE OF INDUCTION OF
AUTOPHAGY. AS WE CAN SEE IN LINE
3 STABILIZING OGT APPEARS TO
ENHANCE CONVERSION OF LC 31 TO
LC 32, SUGGESTING ENHANCING
O-GlcNac LEVELS IS
POTENTIATING AUTOPHAGYPHAGY.
CATALYTICALLY INEFFICIENT SHOWN
IN LANE 5 SHOWS ENEMY AT
INTERMEDIATE PHENOTYPE. WHEN WE
INHIBIT LYSOSOMALATP ACE WITH
BAFLOMYCIN WE SEE ACCUMULATION
AND THIS SUGGESTS THAT
O-GlcNac IS POTENTIATING
THE INDUCTION OF AUTOPHAGY
RATHER THAN BLOCKING FUSION AT
THE AUTOPHAGOSOME AND BLASTSOME.
CANALS PERFORM SIMILAR
EXPERIMENTS USING CELLS
EXPRESSING O-GlcNacASE OR
CAT LICKICALLY INEFFICIENT
O-GlcNacASE. SIMILAR TO
OGT STABILIZATION OF THE
O-GlcNacASE RESULTS IN
ACCUMULATION OF THIS LIPID DATED
FORM OF LC 3 WHEREAS
CATALYTICALLY INEFFICIENT
O-GlcNacASE PHENOCOPIES IT
IS YELLOW FLUORESCENT PROTEIN.
UNLIKE OGT TREATMENT WITH
BAFLOMYCIN RESULTS IN LITTLE
CHANGE IN AMOUNTS OF LIPIDATED
FORM OF LC 3 IN CELLS WHERE THE
O-GlcNacASE HAS BEEN
STABILIZED. WHAT THESE DATA
SUGGEST IS OVER EXPRESSION OF
O-GlcNacASE IS BLOCKING
FUSION OF THE AUTOPHAGOSOME AND
LYSOSOME RATHER THAN
POTENTIATING AUTOPHAGY. SADLY I
DON'T HAVE TIME TO TELL YOU THE
REST OF CANAL'S STORY TODAY BUT
I DID WANT TO SUM WHAT HE
COMPLETED UP. HE'S DEMONSTRATED
THAT AUGMENTING O-GlcNac
LEVELS IN VIVO AND VITRO USING
GENETIC AND FARM O COLLAGECAL
APPROACHES INCREASECREASES AUTOPHAGY
MARKERS AND THIS IS DEPENDENTS
ON AMPK. ELEVATED O-GlcNac
ACTIVATES AMPK AND ULK 1,
AUTOPHAGY BLOCKS ABILITY TO
O-GlcNac TO MEDIATE
CYTOPROTECTION. AMPK IS
GLYCOSYLATED IN RESPONSE TO
OXIDATIVE STRESS AND NOW MAPPED
MORE THAN 12 SITES OF
GLYCOSYLATION IN KEY REGULATORY
MODULES OF AMP KINASE. THANKS
AGAIN TO THE COMMON FUND, IN
SUMMING UP HERE HOW WE HAVE
SHARED OUR REAGENTS, HOW WE
PRESENTED THIS WORK IN A RANGE
OF DIFFERENT SEMINARS AND
WORKSHOPS, AND THAT WE HAVE
TRIED TO VALIDATE AND
DISSEMINATE OUR TOOLS. THANK YOU
FOR YOUR ATTENTION.
>> OUR NEXT SPEAKER ON THIS
SERIES ON O-GlcNac IS DR.
JENNIFER KOHLER.
>> GOOD AFTERNOON, EVERYBODY. AS
I START OFF I WOULD LIKE TO
EXPRESS MY GRATITUDE FOR THE
OPPORTUNITY TO BE PART OF THE
COMMON FUND INITIATIVE. IT'S
BEEN A PLEASURE TO BE PART OF
THIS EFFORT MUCH THERE THAN MY
OWN RESEARCH GROUP AND HAVE A
FRONT ROW SEAT TO DEVELOPMENT OF
NEW TOOLS, ALL NEW TOOLS THAT
ARE GOING TO PAVE THE WAY FOR
THE FUTURE OF GLYCOSCIENCE
RESEARCH.
I WOULD LIKE TO THANK PAM,
AMANDA AND CARL FOR LEADING THE
WAY AND ALSO THANK CATHERINE
GRIMES FOR HER LEADERSHIP ON THE
TOOLS SUBGROUP. FOR OUR COMMON
FUND PROJECT WE -- I DECIDED
THAT IT WOULD BE A GREAT
OPPORTUNITY TO ENGAGE SEVERAL OF
MY COLLEAGUES FROM UT SOUTH
WESTERN WHO ARE NOT NECESSARILY
INTERESTED IN GLYCOSCIENCE BUT
HAVE EXPERTISE THAT ENABLES
GLYCOSCIENCE TOOLS SO THE WORK I
WILL TELL YOU ABOUT INCLUDES
COLLABORATIONS WITH MY
COLLEAGUES NICK CONRAD
DEPARTMENT OF MICROBIOLOGY,
UTTAM TAMBAR FROM SAME
DEPARTMENT AS ME BIOCHEMISTRY
AND LUKASZ JOACHIMIAK FROM THE
ALZHEIMER'S CENTER. THANK YOU.
SO THE FIRST THING I WILL TALK
TO YOU ABOUT IS THE PROJECT THAT
WAS CONDUCTED IN NICK'S LAB AND
THIS IS AIMED AT DEVELOPING THE
PORTER TO TELL US SOMETHING
ABOUT O-GlcNac LEVELS
INSIDE CELLS OR POTENTIALLY
INSIDE ORGANISMS. THIS REPORTER
WAS BASED ON OBSERVATION THAT
NICK CONRAD'S LAB MADE A FEW
YEARS AGO REGARDING THE SPLICING
OF THE TRANSCRIPT OF OGT,
PROTEIN THAT TRANSCRIPT THAT
ENCODES OGT PROTEIN,
O-GlcNac TRANSFERASE. AND
IN THIS CASE'S LAB IIN THISNICK'S LAB IS I NTERESTED IN
SPLICING AND DETAIN INTRON SOP
WHERE INTRONS ARE NOT
EFFICIENTLY SPLICED OUT OR
REGULATED -- REGULATORY
MECHANISM FOR SPLICING, THEY
DISCOVERED ONE INTRON IN THE OTT
TRANSCRIPT WAS SPLICED OUT IN
O-GlcNac DEPENDENT WAY SO
UNDER CONDITIONS OF LOW
O-GlcNac WITHIN THE CELL,
THIS TRANSCRIPT HOE SUFFICIENTLY
SPLICED LEADING TO OGT
TRANSCRIPT AND PROTEIN WHICH
ALLOWS INSTALLATIONS OF
ADDITIONAL O-GlcNac TO
RESTORE HOMEOSTASIS
O-GlcNac LEVELS, HOWEVER,
UNDER CONDITIONS OF HIGH
O-GlcNac, THIS INTRON IS
RETAINED AND THE TRANSCRIPT IS
DEGRADED AND THERE'S LESS OGT
AVAILABLE. SO NICK'S LAB THOUGHT
IT WOULD BE POSSIBLE TO THEN USE
THAT SAME INTRON RETENTION
MECHANISM TO GENERATE
O-GlcNac RESPONSIVE
REPORTERS AND THE IDEA HERE WAS
TO INCORPORATE THE GF -- GENE
ENCODING GFP ON TO THE SAME
TRANSCRIPT AND SO IN THIS WAY
THE PRODUCTION OF FLUORESCENT
PROTEIN WOULD NOW BE SENSITIVE
TO O-GlcNac LEVELS. SO
THEY DESIGNED A CONSTRUCT TO DO
THIS SO UNDER CONDITIONS OF LOW
O-GlcNac THIS TRANSCRIPT
IS SUFFICIENTLY PROCESSED,
PRODUCING GFP AND UNDER
CONDITIONS OF HIGH
O-GlcNac THE TRANSCRIPT IS
DEGRADED AND THERE'S LESS GFP.
SO THE LEVELS IS INVERSELY
PROPORTIONAL TO THE LEVEL OF
O-GlcNac. IN THE CELLS.
THEY DEVELOP THIS CONSTRUCT
EXPRESSED IT SABLELY AND
MAMMALIAN CELLS AND SHOW INDEED
IT IS RESPONSIVE TO
PERTURBATIONS THAT CHANGE
O-GlcNac LEVELS IN CELLS.
THIS IS SHOWN BY WESTERN BLOT
USING ANTIBODY AGAINST
O-GlcNac MODIFICATION AND
ALSO BY FLOW CYTOMETRY WHERE WE
CAN SEE THE LEVELS OF THE GFP
CHANGING IN RESPONSE TO
INHIBITORS SO THIAMET G INHIBITS
OGA AND RAISES O-GlcNac
LEVELS AND WE SEE LESS GFP
FLUORESCENCE BY FLOW CYTOMETRY,
WHEREAS OSMI 1 INHIBITS OGT
RESULTING IN LOYER
O-GlcNac LEVELS AND HIGHER
FLUORESCENCE. NICK'S LAB ALSO
VALIDATED THIS BY MICROSCOPY
WHICH IS SHOWN HERE, AND IN
WHERE THEY KNOCK DOWN OGT
TRANSCRIPT USING SI RNA AND YOU
CAN SEE AN INCREASE IN THE
FLUORESCENCE BY MICROSCOPY AND
FLOW CYTOMETRY. SO THIS TOOL IS
KIND OF READY TO BE USED IN
DIFFERENT WAYS AND SO YOU CAN
IMAGINE YOU MIGHT DO SOME
PERTURBATION TO THE CELL AND
WANT TO KNOW IF THERE IS CHANGE
IN O-GlcNac LEVELS AND ONE
COULD USE THIS REPORTER IN THIS
WAY. NICK'S LAB IS USING THIS
TO STUDY THE SPLICING OF OGT
TRANSCRIPT AND THEY INTRODUCED
THIS REPORTER, AND USED A
CRISPER LIBRARY AND NOW LOOKING
-- THEY FOUND WHICH GENES MODIFY
THIS RESPONSE. SO THEY TRYING TO
VALIDATE AND DETERMINE EXACTLY
WHICH SPLICE FACTORS ARE
INVOLVED IN THE SENSING OF
O-GlcNac TO REGULATE THE
SPLICING OF OGT TRANSCRIPT. THEY
ARE ALSO PRODUCING ADDITIONAL
REPORTER IN WHICH LUCIFERASE IS
PRODUCED FROM GFP AND ONE CAN
IMAGINE USING THAT IN ANIMALS.
SO IF PEOPLE ARE INTERESTED IN
USING THIS THEY ARE WELCOME TO
CONTACT NICK OR ME BECAUSE WE
THINK THIS REPORTER COULD BE
USED IN A LOT OF WAYS. ALL
RIGHT. NEXT KIND OF PARTS OF OUR
PROJECT WERE FOCUSED ON
UNDERSTANDING THE INTERACTION
PARTNERS OF GLYCOSYLATED
MOLECULES AND THIS CARTOON HERE
GIVES THE BIG PICTURE WHAT WE
ARE THINKING ABOUT. AND THE IDEA
IS THAT PROTEINS THAT ARE
GLYCOSYLATED IN SPECIFICALLY IN
THIS CASE O-GlcNac LATED
YOU CAN SEE THIS IN TERMS OF
PERHAPS WHERE THEY LOCALIZE IN
THE CELLS OR BINDING PARTNERS OR
OTHER PROTEINS THAT ARE NEARBY,
ONE CAN IMAGINE YOU CAN LEARN
ABOUT THE PROTEINS THAT THEY
HANG OUT WITH YOU LEARN
SOMETHING ABOUT WHAT THEY ARE
DOING. SO THAT WAS THE IDEA
BEHIND THIS. A FEW YEARS AGO WE
DECIDED TO APPROACH THIS PROBLEM
BY INCORPORATING A PHOTO
ACTIVATABLE CROSS LINKER ON TO
THE O-GlcNac LEVELS IN
LIVING CELLS. . WE DID THIS BY
-- I WILL DO WITHOUT THE
POINTER. THE WAY WE DID THIS WAS
BY PRODUCING THE COMPOUNDS SHOWN
IN THE UPPER LEFT DIAZIRINE
PHOTOCROSS LINKER GLOMERULAR
ANALOG AND PROTECTING GROUPS ARE
IN PURPLE. THESE PROTECTING
GROUPS ALLOWED US TO ENTER THE
CELL, REMOVE THE INTRACELLULARLY
REVEALING THE GLCNDAZ 1
PHOSPHATE. TO ACTIVATE THIS IN
UDP SUGAR IN CELLS WE IMMEDIATED
TO INTRODUCE MUTANT ENZYME
NATASHA POINTED OUT IN A EARLIER
TALK SO A MUTATION TO THE
O-GlcNac PHOSPHORYLATES
THAT ALLOWS OUTS TO ACCEPT THE
DIAZIRINE MODIFIED COMPOUND.
ONCE CELLS MAKE THIS MODIFIED
UDP QUICK KNACK ANALOG WITH
KAIZARINE IT IS A SUBSTRATE AND
CAN CROSS LINK O-GlcNac
PROTEINS TO THEIR NEIGHBORS
WITHIN LIVING CELLS. SO IN OUR
COMMON FUND PROJECT WE AIM TO
IMPROVE AND EXPAND THIS
TECHNOLOGY AND ONE THING THAT
WAS A PROBLEM REALLY WAS MAKING
THAT MOLECULE SHOWN AT THE UPPER
LEFT AND SO WE HAD A STRATEGY TO
MAKE IT BUT IT WAS I WOULD SAY
QUITE UNRELIABLE AND WE KNEW
FROM OTHERS AS WELL MAKING THIS
PROTECTIVE PHOSPHOSUGAR WE SAW
VARIABLE YIELDS BASICALLY. AND
SO MY COLLEAGUE UTTON TAMBAR
DIVIDESSED A MORE RELIABLE
APPROACH TO GET TO THIS MOLECULE
WHICH WE USE COMMERCIAL REAGENT
SHOWN IN UPPER LEFT IN RED TO
MAKE THE INITIAL PHOSPHOSUGAR
AND THEN EXCHANGE IN THE DIASTE
ARE PROTECTING GROUPS ON
PHOSPHATE AND FINAL OXIDATION
STEP. SO THIS GRAPH TO THE
COMPOUND IS MORE RELIABLE AND
MORE SCALABLE AND ALLOWS ACCESS
TO BOTH ORIGINAL MOLECULE WE
HAVE BEEN USING QUITE SOME TIME
AS WELL AS ADDITIONAL ANALOGS.
AND SO WE PREPARED THIS GLCDAZ 1
PHOSPHATE BUT THE GALNAC AS WELL
AS OTHER ANALOGS WE REPLACE
DIAZIRINE WITH DIFFERENT
FUNCTIONAL GROUPS SO WE CONTINUE
TOE VALUE WAIT ADDITIONAL
ANALOGS. -- EVALUATE THE
ADDITIONAL ANALOGS. THE OTHER
WAY TO EXPAND THE TECHNOLOGY IS
TESTING WHETHER THE PHOTOCROSS
LINKING GLCNAC WAS
PHOSPHORYLATED INTO
GLYCOCONJUGATES. THIS WAS DONE
IN COLLABORATION WITH SEVERAL --
TWO GROUPS, AT THE CCRC,
(INAUDIBLE) GROUP WITH MASS
SPECTROMETRY ANALYSIS AND KELLY
KELLY'S GOSH WITH IN VITRO
GLCNAC TRANSFERASE ACTIVITIES
AND THEY DID WORK SUMMARIZED IN
THIS ONE SLIDE THAT ALLOWED US
TO CHARACTERIZE HOW THELCNGZAZ
WAS INCORPORATED INTO N LINKED
GLYCANS. WE ONLY SAW ONE
INCORPORATED PER LINK GLYCAN, IT
WAS KNOCKED DOWN IN CORE
POSITIONS, IT WAS WE BELIEVE
LIKELY INCORPORATED BY NGAP 2,
POTENTIALLY ALSO INCORPORATED BY
NGAP 5 AND IMPORTANTLY THESE
GLCNDAZ INCORPORATED EXTENDED
WITH GAU THAT ALLOWS FORMATION
OF MATURE GLYCANS AND LINK
GLYCANS. SO ONCE WE KNEW THAT
GLCNDAZ IS GLYCOSYLATED INTO
GLYCANS IN MY LAB USED THIS TOOL
TO LOOK AT N LINK GLYCAN BINDING
INTERACTIONS AND SPECIFICALLY WE
USED GALECTIN 1 AND WE ADDED
THIS EXTRA CELLULARLY TO CELLS
THAT WERE MAKING THESE MODIFIED
IN LINK GLYCANS WITH CROSS
LINKER AND WE COULD OBSERVE THEN
WITH UV RADIATION OBSERVE CROSS
LINKING OF GALECTIN 1 TO CELL
SURFACE GLYCOPROTEINS P AND BY
USING BIATEN LATED FORM YOU CAN
PULL OUT PROTEINS AND IDENTIFY
BY MASS SPEC AND FOUND INDEED
MANY ARE ANNOTATED AS N LINK
GLYCOPROTEINS AND SOME KNOWN TO
BE BINDING PARTNERS OF GALECTIN
1 PREVIOUSLY REPORTED IN THE
LITERATURE. SO IN FACT WE DID
THIS ANALYSIS IN CD4 CELLS AND
QUITE SOME TIME AGO IN THE '
'90s THERE WAS A REPORT THAT
GALECTIN 1 WAS USED FOR AFFINITY
PURIFICATION METHOD TO IDENTIFY
N LINK GLYCOPROTEINS FROM CELL
LINES AND TWO PROTEINS
IDENTIFIED WERE CCAM 5 AND LAMP
1 SO WE DECIDED TO TEST TO SEE
IF WE CAN OBSERVE THAT CROSS
LINKING OF GALECTIN 1 TO TWO
PROTEINS IN OUR CELLS, THIS
INVOLVES ENINCORPORATING THE
GLCNGDAZ MEDIATED TO CROSS LINK
AND ACROSS PROTEIN OF INTEREST
CCAN 5 WITH LAMP 1 AND GALECTIN
1 TO LOOK FOR COMPLEX IN THE
GEL. YOU CAN SEE THE GEL ON THE
LEFT WE CAN OBSERVE CROSS
LINKING OF CCAM 5 TO GALECTIN 1
BOTH IN INTACT CELLS BUT ALSO
WHEN THE CROSS LINKING WAS
PERFORMED AFTER WE FIRST LYSED
THE CELLS, AND PERFORMED CROSS
LINKING IT WAS THE SAME NO
MATTER EXPERIMENT. BUT DOING
WITH LAMP 1 WE DID NOT OBSERVE
CROSS LINKING WITH INTACT CELLS
BUT ONLY WHEN WE FIRST LYSE THE
CELLS AND DID CROSS LINKING.
THAT WAS INTERESTING BECAUSE
LAMP 1 DIDN'T SHOW IN PROTEOMICS
ANALYSIS WHEN GALECTIN WAS A
APPLIED EXTRA CELLULARLY SO
SHOWS THE BENEFIT OF CROSS
LINKING METHOD IS TELLS YOU
ABOUT THE INTERACTIONS THAT ARE
OCCURRING IN LIVING CELLS, AND
WHEREAS AFFINITY PURIFICATION
SCRAMBLES THE INTERACTIONS SO
YOU MAY INTRODUCE SPURIOUS
FINDING INTERACTIONS IN THAT
WAY. SO WITH THAT OBSERVATION WE
WENT TO LOOK AT THE CROSS
LINKING OF ENDOGENOUS GALECTIN 1
INSIDE THESE CELLS AND WHAT IS
INTERESTING IS IF WE DON'T TREAT
THE CELLS WE OBSERVE NO CROSS
LINKING OF GALECTIN 1 ENDOGENOUS
GALECTIN 1 INSIDE CELLS BUT IF
WE TREAT WITH A LYSOSOMAL DAMAGE
REAGENT, WHICH EXPOSES THE
CONTENT OF THE LYSOSOME WHICH
INCLUDES N LINK GLYCOPROTEINS TO
GALECTINS WHICH ARE IN CYTOPLASM
WE OBSERVE CROSS LINKING OF
GALECTIN 1 TO PROTEINS WE ASSUME
ARE LYSESOMAL PROTEINS. THAT IS
INHIBITED WE USE INHIBITOR OF
LYSOSOMAL DAMAGE PATHWAY. SO WE
ARE CONTINUING TO FOLLOW-UP ON
THIS TO UNDERSTAND EXACTLY HOW
GALECTINS INTERACT WITH
LYSOSOMAL PROTEINS AND
CONDITIONS OF LYSOSOMAL DAMAGE.
SO JUST TO CONCLUDE THIS PART
ABOUT INCORPORATION OF GLCNDAZ
TO N LINKED PROTEINS, IT WAS
METABOLICALLY INCORPORATED TO N
LINK GLYCANS. WE CAN LEARN
ABOUT THE AMOUNT OF THESE
BINDING EVENTS THROUGH THE
INTENSITY OF THE CROSS LINKING,
WE CAN ISOLATE CROSS LINK
COMPLEXES AND MOST IMPORTANTLY
GIVES US CELLS CONTEXT SPECIFIC
INFORMATION ABOUT RECOGNITION
EVENTS. WORKING WITH (INAUDIBLE)
GROUP WE ARE CONTINUING TO
EVALUATE INCORPORATION OF
GLCNDAZ INTO OTHER CLASSES OF
GLYCOCONJUGATES. I WANT TO TELL
YOU ABOUT THE LAST AIM WHICH IS
STILL UNDERWAY, AND THIS WAS --
WE OUR AIM WAS TO CHARACTERIZE
IN MORE DETAIL SITES WHICH THE
CROSS LINKING EVENTS OCCUR AND
THIS WAS WE DECIDED TO GO ABOUT
THIS BY LOOKING AT A MODEL
SYSTEM AND RECOGNITION OF
O-GlcNac PEPTIDE BY
PROTEIN 14-3-3 GAMMA, MY LAB
IDENTIFIED THE INTERACTION AND
SOLVED THE CRYSTAL STRUCTURE OF
BINDING INTERACTION A FEW YEARS
AGO SO WE KNEW WHERE THE GLCNAC
CONTACT THE 14, 3, 3 PROTEIN. WE
PREPARED A POPE TIDE WITH
OGLCNDAZ ON IT AND CROSS LINKED
THE COMPLEX AND TRYPSIN DIGEST
IT AND USED THE BIONIC SOFTWARE
ALSO SUPPORTED THROUGH THE
COMMON FUND AND SHARED WITH US
THROUGH THAT EFFORT TO LOOK FOR
ADDUCTS THAT REPRESENT CROSSING
OF PEPTIDE TO 14-3-3 PROTEIN.
WESTERN ABLE TO FIND ONE SPECIES
THAT CORRESPONDED TO THIS, WE
ARE LOOKING TO LOOK AT OTHER
MODEL SYSTEMS TO SEE IF WE CAN
SEE THIS KIND OF CROSS LINKING
EVENTS IN SYSTEMS WHERE WE DON'T
HAVE STRUCTURAL INFORMATION. SO
THAT WORK IS UNDERWAY. ALL
RIGHT. SO THIS IS MY SUMMARY OF
OUR ENTIRE PROJECT WE PUT
TOGETHER. THE CONRAD LAB CREATE
AD FLUORESCE SENT REPORTER OF
O-GlcNac LEVELS BASED ON
THE SPLICING OF OGT TRANSCRIPT.
WE DEVELOPED A MORE RELIABLE
SYNTHETIC ROUTE TO MAKE HEXOSE
MIEN 1 PHOSPHATE ANALOGS USING
FOR OTHER COMPOUNDS TO FIND
SUGARS WE WERE INTERESTED IN AND
WE DEMONSTRATED THE GLCNDAZ
COMPOUND IS INCORPORATED TO N
LINK GLYCANS AND USEFUL TO PROBE
INTERACTIONS. WE ARE CONTINUING
TO INCORPORATE EVALUATE
INCORPORATION OF THESE UNNATURAL
SUGARS AND WRAPPING UP THAT MASS
SPEC ANALYSIS OF THE SITE TO
CROSS LINKING. SO I WANT TO WRAP
UP WITH A COUPLE OF WORDS ABOUT
DISSEMINATION. WE WERE THROUGH
THE EFFORTS OF CATHERINE WE WERE
ABLE TO PUT TOGETHER THIS
CURRENT PROTOCOLS TO SHARE IN
CURRENT PROTOCOLS. OUR REAGENT
TO INTRODUCE UNNATURAL UAP IS
AVAILABLE FROM ADD GENE AND YOU
HEARD THERE IS REAGENTS FROM
NATASHA'S LAB AS WELL. FOR NOW
YOU CAN REQUEST COMPOUND FROM
US, BUT CHRIS IS WORKING ON
PREPARING FOR COMMERCIAL
AVAILABILITY. TOCRIS. THESE ARE
SOME OF THE PUBLICATIONS WE HAVE
HAD AND I WOULD LIKE TO THANK
ALL MY COLLABORATORS BOTH AT UT
SOUTH WESTERN AND AT UNIVERSITY
OF GEORGIA, IT'S BEEN GREAT TO
WORK ON THIS PROJECT WITH ALL OF
THEM. THANK YOU FOR YOUR
ATTENTION.
[APPLAUSE]
>> OUR NEXT SPEAKER IS DR.
CHRISTINE WOO, HARVARD. AS HAS
BEEN BEAUTIFULLY INTRODUCED BY
NATASHA NOW JEN THIS
CARBOHYDRATE HERE HOLDS
O-GlcNac, IS FOUND ON
SERINE 3 ANINE RESIDUES WITHIN
THE CELL NUCLEAR CYTOPLASMIC AND
MITOCHONDRIAL PROTEINS. IT IS A
METABOLIC SENSOR THAT IS QUITE
IMPORTANT AND I WON'T REITERATE
ALL THE DETAILS OF WHY YOU
SHOULD THINK ABOUT THIS SUGAR
MOLECULE, BUT THE KEY PART THAT
FASCINATED US IS THAT THIS SMALL
MOLECULE IS GLYCOSYLATION EVENT
OCCURS ON ALL THESE PROTEINS
SUBSTRATES WITH THE ACTIVITY OF
A SINGLE SET OF ENZYMES, WRITER
O-GlcNac TRANSFER RACE OR
OGT AND ONE ERASER OGA. THERE'S
DYNAMICS WEAN THE TWO ENZYMES
THAT IS REGULATING THE LEVELS OF
THIS SUGAR. SO THESE TWO ENZYMES
ARE QUITE ESSENTIAL, THEY ARE
REQUIRED FOR CELLULAR DIVISION
AND CELLULAR PROLIFERATION. THEY
ALSO STABILIZE NUMBER OF
PROTEINS IN OUR CELLS INCLUDING
ALPHA SYNUCLEIN, WHICH WHEN
ALPHA SYNUCLEIN IS GLYCOSYLATED
IT IS SOLUBLE BUT WHEN
DEGLYCOSYLATED IT FORMS
AGGREGATES AND AFTER LUNCH AS WE
ALL FEEL SATIATED, THAT IS
PLAYING O-GlcNac IS
PLAYING A ROLE IN OUR BRAINS AND
TELLING US THAT WE HAVE ENOUGH
SUGAR AND ENERGY AND IF WE LOSE
OGT IN CERTAIN PARTS OF THE
BRAIN THIS CAN INDUCE DIABETIC
PHENOTYPE AND NOT BEING ABLE TO
SIGNAL WHEN HOW MUCH FOOD IS
AVAILABLE. SO WHAT IS QUITE
INTERESTING TO US AND I THINK
REALLY WITH INNOVATIONS WITH
ASSISTANCE OF INNOVATIONS ON
MASS SPEC THAT WE WILL HEAR
ABOUT IN THE COMING DAY OR TWO,
HAS BEEN NOW THAT WE HAVE THESE
DEEPER DEEPER MAPS OF WHERE AND
WHEN O-GlcNac IS OCCURRING
BY MASS SPEC WE NOW KNOW THIS
SUGAR MOLECULE IS FOUND ON OVER
5,000 DIFFERENT PROTEINS. MANY
OF WHICH SPAN NUMBER OF
SIGNALING PROTEINS LIKE
TRANSCRIPTION FACTORS, RNA
BINDING PROTEINS, KINASE AND E 3
LIGASE. SO CENTRAL QUESTION WE
STARTED TO ASK IN MY LAB IS HOW
DOES O-GlcNac TAYLOR
PROTEIN FUNCTIONS IN CELLS?
SECONDARILY WE CAN ANSWER THE
QUESTION HOW AND WHERE TO USE
O-GlcNac TO MITIGATE HUMAN
DISEASES. SO AS WE CONTEMPLATED
THIS QUESTION AND HOW WE CAN
ANSWER IT, WE REALIZE WE NEED AD
BETTER WAY TO ACTUALLY
MANIPULATE O-GlcNac IN A
CELLULAR CONTEXT ABLE TO ASK HOW
O-GlcNac FUNCTIONS AND
THEN HOW WE CAN USE IT FOR
BENEFICIAL INTERCEPTION. AS WE
THOUGHT ABOUT DIFFERENT OPTIONS
FOR USING MANIPULATING
O-GlcNac, WE CAME UP WITH
THIS IDEA WE THOUGHT WELL, IF
ONLY WE COULD TAKE A TARGET
PROTEIN AND RECRUIT
O-GlcNac TO IT AND IN THIS
WAY BY RECRUITING THAT
PROXIMITY, THAT O-GlcNac
TRANSFERASE THROUGH PROXIMITY
ENHANCING
O-GglcNaCYLATION ON THAT
PROTEIN. CONTRAST IF WE REDUCE
THE SINGER E RAZOR
O-GlcNacASE WE CAN
DEGLYCOSYLATE IT. AND IF WE CAN
CYCLE SELECTIVELY ON TARGET
PROTEIN IN CELLULAR CONTEXT WE
CAN CHARACTERIZE RULE OF THAT
GLYCOSYLATION EVENT EVENT. AS WE
THOUGHT ABOUT WAYS TO RECRUIT
AND RECOGNIZE TARGET PROTEINS,
IT BECAME -- OBVIOUSLY CELLS
KNOW HOW TO DO THIS AND THEY
KNOW HOW TO DO IT USING ANTIBODY
SO ANTIBODIES ARE HIGHLY
SPECIALIZED PROTEIN RECOGNITION
SYSTEMS BUT PRIMARILY FOUND ON
THE OUTSIDE OF CELLS, SO
ANTIBODIES RECOGNIZE NUMBER OF
PROTEINS WITH REALLY HIGH
SELECTIVITY AND SENSITIVITY ON
CELL SURFACE. THIS DOESN'T BODE
WELL FOR INTRACELLULAR PROTEINS,
BUT FORTUNATELY THERE IS
EMERGING RESEARCH IN THE AREA OF
NANOBODIES AND OTHER SINGLE
DOMAIN PROTEIN BINDERS, WHICH WE
ARE PARTICULARLY ATTRACTED TO
NANOBODIES, THEY ARE ESSENTIALLY
SMALL ANTIBODIES THAT
SPECIFICALLY DERIVE FROM CAMELS.
AND THESE SMALL ANTIBODIES ARE
ESSENTIALLY HIGH AFFINITY
PROTEIN RECOGNITION SYSTEMS THAT
ARE NOW ALSO SMALL SINGLE DOMAIN
PROTEIN BINDERS THAT WE CAN FUSE
TO THE O-GlcNac TRANSFER
TRANSFERRIST AND
O-GlcNacASE. SO I WILL
TELL YOU ABOUT THE DESIGN OF THE
SYSTEM AND HOW WE USE IT TO DATE
AND DISSEMINATION AND FUTURE
PLANS. SO IN TERMS OF DESIGNING
THE WRITER TARGET PROTEIN
WRITER, OF O-GlcNac, WE
FIRST LOOK AT STRUCTURE OF
O-GlcNac TRANSFERASE WE
ARE SHOWING YOU LINEAR STRUCTURE
AS AS WELL AS CRYSTAL STRUCTURE
DOWN HERE. SO O-GlcNac
TRANSFERASE HAS TWO PRIMARY
DOMAINS, ONE IS CATALYTIC DOMAIN
SHOWN HERE IN YELLOW AND THE
OTHER PART IS THIS HETERO
PEPTIDE REPEAT DOMAIN IN PURPLE.
THE CATALYTIC DOMAIN WHERE ALL
THE ACTION IS HAPPENING FOR THE
GLYCOSYLATION EVENT IS
HIGHLIGHTED HERE IN SPECIFIC
AMINO ACID RESIDUES IN RED. ALL
THAT ACTION IS SHOWN HERE BUT
HOW O-GlcNac RELATION
EVENTS OCCUR IS POSITIONED ALL
BY THE TETRA TRICO PEPTIDE
REPEAT DOMAIN AND CONTACTS
THROUGHOUT THIS DOMAIN. WHAT WE
ENVISION IS PERHAPS REDUCE AND
REMOVE THIS DOMAIN AND REP PLACE
WITH A NANOBODY AND HOW
O-GlcNac TRANSFER AIS IS
DOING PROXIMITY INDUCTION IN
DOMAIN AND REPLACING SUBSTITUTE
WITH ANTIBODY. SO THAT IS WHAT
WE DID. WE TOOK THAT TPR DOMAIN
AND TURNS OUT YOU CAN'T GET RID
OF IT COMPLETELY, YOU CAN GET IT
DOWN TO ABOUT FOUR TETRATRICO
PEPTIDE REPIECE AND GET MOST
CONSENSUS SEQUENCE BUT REDUCE
BACKGROUND AND LIMIT ENZYME
ABILITY TO EFFECTIVE BROAD SWATH
OF SUBSTRATES, AND THEN
SUBSTITUTE THAT WITH NANOO BODY.
SO AGAIN THE IDEA IS THAT WE
HAVE THIS TARGET PROTEIN WE ARE
INTERESTED IN, AND HAPPENS TO
HAVE A TAG ON IT, WE CAN RECRUIT
THAT THROUGH THE NANOBODY OR IF
WE HAPPEN TO HAVE ONE THAT
RECOGNIZES ENDOGENOUS TARGET
PROTEIN WE CAN RECRUIT IT TO
THAT TARGET PROTEIN AS WELL.
WITHOUT AFFECTING BROADER
GLYCOPROTEOME. SO SOME OF OUR
FAVORITE NANOBODIES ARE SHOWN
HERE INCLUDING NANOBODIES
AGAINST GFP SEVERAL AFFINITIES,
NANOBODIES THAT DIRECTLY
RECOGNIZE PROTEINS LIKE ALPHA
SYNUCLEIN AND OTHER PEPTIDES
TABS 6 PEPTIDE OR BC 2 PEPTIDE.
THIS WORK WAS ESTABLISHED BY ONE
OF MY FIRST GRAD STUDENTS NOW
GRADUATED AT STANFORD IN
POST-DOCTORAL FELLOWSHIP DANIEL
RAMMIER RUIZ. SO YOU TAKE THE
NANOBODY AND INTRODUCE TO CELLS
LINES FOR TRANSFECTION AND
CHARACTERIZE O-GlcNacATEED
PROTEOME SO THIS IS A WRITER OF
O-GlcNac WE NEED AN ERASER
TO ERASE THE MODIFICATION. NOW
INSTEAD OF RECRUITING
O-GlcNac RELATION WE WANT
TO REMOVE IT. FORTUNATELY AS WE
STARTED TO THINK ABOUT THIS
SEVERAL CRYSTAL STRUCTURE OF OGA
WERE PUBLISHED AND WHEN WE LOOK
AD IF THE STRUCTURE AND THE
RELATIONSHIP WORK THEY DID WAS
REALLY BEAUTIFUL ABOUT THE OGA
STRUCTURES, ACTUALLY A
HOMODIMER, WITH ONE EQUIVALENT
OF THE OGA SHOWN HERE IN DARK
RED AND DARK BLUE AND ANOTHER IN
LIGHT BLUE AND LIGHT PINK.
CORRESPONDING TO THE CATALYTIC
DOMAIN IN PINK AND THE STOCK
DOMAIN SHOWN IN BLUE. ONE OF THE
REALIZATION IS THAT WE HAD FROM
LOOKING AT THIS STRUCTURE AND
FROM RECENTLY PUBLISHED WORK WAS
THAT PERHAPS WE COULD AGAIN
REMOVE REDUCE SOME OF THE
ACTIVITY OF THE ENZYME AND
REPLACE WITH NANOBODY, IN THIS
CASE WE CAPITALIZE ON A SPECIFIC
NATURAL CLEAVAGE EVENT FOUND
WITHIN OGA SHOWN HERE THAT
SEPARATES THE CATALYTIC DOMAIN
FROM STOCK DOMAIN REDUCING THE
FORMATION OF THIS CALLED
PLAQUES. UNFORTUNATELY WE CAN
THEN -- WE USING A SYSTEMATIC
ENGINEERING APPROACH REALLY
PIONEERED AND HERALDED BY YUN GE
FORMER POST-DOC IN MY LAB NOW AT
BAY LABORATORY IN HER OWN
INDEPENDENT GROUP REALLY
SYSTEMATICALLY ENGINEERED A
SPLIT OGA SYSTEM CONSISTING OF
THE CATALYTIC DOMAIN WITH THE
STALK DOMAIN AS INDEPENDENT
FRAGMENT. THIS SYSTEM ON ITS OWN
HAS LIMITED CATALYTIC ACTIVITY
ON ENDOGENOUS SUBSTRATES AND
ONLY WHEN WE INTRODUCED THE
NANOBODY TO THAT STALK DOMAIN
CAN WE RECOVER THE ACTIVITY. SO
IN AIM ONE WAS FOCUSED ON
WRITING AND SYSTEMATICALLY
CHARACTERIZING WITH NANOBODIES
AND TWO PROPALE WAS LOOKING AT
ERASING THE MODIFICATION AND NOW
THAT WE HAVE THESE TOOL SYSTEMS
WE CAN USE THEM FOR
CHARACTERIZING
O-GlcNaCYLATION EVENTS AS
TOOLS. IF YOU WANT YOUR OWN
NANOBODY INFUSED ENZYMES FOR
OTHER GLYCOSYLATION EVENTS WE
LEARNED THROUGH OUR WORK THAT
THIS SYSTEMATIC APPROACH FIRST
REDUCING ACTIVITY OF THE ENZYMES
ENDOGENOUS SUBSTRATE RECOGNITION
MECHANISMS, BY WHICH YOU CAN
LEARN ABOUT THE STRUCTURE
ACTIVITY RELATIONSHIPS OF THESE
ENZYME SYSTEMS, TO LIMIT THE
BACKGROUND AND FOLLOWED BY
RECOVERING THE ACTIVITY WITH
NANOBODY CAN GIVE YOU SELECTIVE
SYSTEMS. IN THE LAST FEW MINUTES
I WANT TO SHARE A KEY QUESTION I
THINK THAT MAJOR MOTIVATION OF
THE COMMON FUND AND OURS AS WELL
IS HOW I CAN USE THE
O-GlcNac WRITER AND ERASER
ON MY TARGET PROTEIN. SO I
WANTED TO TELL ONE REALLY BRIEF
VIGNETTE PIONEERED HERE BY PAUL
STEIN ANOTHER SOON TO BE
GRADUATED NOW DR. PAUL SCHWEIN
STUDENT MANY MY LAB. PAUL IS
INTERESTED IN CHARACTERIZING THE
INTERPLAY WITH O-GlcNac
AND PHOSPHORYLATION SPECIFICALLY
THROUGH HOW O-GlcNac CAN
EFFECT KINASES DIRECTLY ON THE
ENZYMES THAT WRITE
PHOSPHORYLATION. SO HE PICKED UP
ON PRECEDENT OUT OF THE COLE LAB
WHERE WHO HAD SHOWN THAT CASEIN
KINASE 2 UBIQUITOUSLY EXPRESSED
ONE OF THE FIRST KINASES
DISCOVERED, UBIQUITOUSLY
EXPRESSED AND ACTIVE WITH
HUNDREDS OF DIFFERENT
SUBSTRATES. THIS KINASE HAS A
SINGLE GLYCOCYTE THAT IN VITRO
SHOWS PROTEIN PROTEIN
INTERACTIONS AS WELL AS
SUBSTRATES AND PAUL WANTED TO
USE THE TOOLS WE DEVELOPED HERE
AS PARTS OF THE COMMON FUND TO
CHARACTERIZE WHETHER OR NOT THIS
SINGLE S 347 GLYCOSIDE ALTERS CK
2 SUBSTRATES. SO THE IDEA TO
USE THESE WRITERS AND ERASERS IS
YOU CAN APPLY THE NANOBODY OGT
SYSTEM TO INTRODUCE THE
GLYCOSYLATION EVENT, HERE
THROUGH A GFP TAGGED CP 2 ALPHA.
AND YOU CAN APPLY NANOBODY OGA
SYSTEM TO REDUCE IT. THE
IMPORTANTLY FOR EVERY ACTIVE
ENZYME WE HAVE AN INACTIVE
ENZYME AS CONTROL. TO OVERCOME
ANY ARTIFACTS FROM THE NANOBODY
RECOGNITION ITSELF. SO PAUL
SHOWED THAT IF HE HAS THAT CK 2
ALPHA SOURCES CELLS AND PRESENCE
ACTIVE NANOBODY OGT WE CAN SEE
ELEVATED O-GlcNaCYLATION
AND THAT IS REDUCED AND REMOVED
OR DOESN'T OCCUR IN PRESENCE OF
THE INACTIVE NANOBODY OGA. IF WE
GLOBALLY ALTER THE
O-GlcNac PROTEOME WE DON'T
SEE AS SIGNIFICANT A INCREASE IN
O-GlcNaCYLATION OF THIS
TARGET PROTEIN COMPARED TO THE
NANOBE DI SYSTEM. BY CONTRAST
AND NOW IF WE COME IN AND LOAD A
LOT MORE PROTEINS WE CAN SEE
O-GlcNaCYLATION
ENDOGENOUSLY AS EXPECTED,
REMOVED BY GLOBAL
DEGLYCOSYLATION AND NOW MORE
TARGETED WAY WITH NANOBODY SPLIT
OGA SYSTEM THAT IS CONTROLLED
WITH THE CATALYTICALLY INACTIVE
VERSION. I DON'T HAVE TIME TO
SHOW ALL THE DATA OF PAUL'S
STUDY BUT HE SHOWED IN ADDITION
THE NANOBODY OGT SYSTEM IS
SELECTIVELY GLYCOSYLATING THE
SINGLE KNOWN GLYCOSIDE AND NOT
HITTING ANOTHER OFF TARGET
O-GglcNaCYLATION EVENT
IN THIS PARTICULAR PROTEIN, HE
CAN KNOCK IT OUT SELECTIVELY
WITH A SINGLE POINT MUTATION OR
A DOUBLE POINT MUTATION. AND
INTERESTINGLY IF WE TRY TO MAKE
A STABLE CYSTEINE GLYCOSYLATION
EVENT ENDOGENOUS OGA IS NOT
AFFECTING THAT. SO REALLY THIS
NANOBODY OGT OGA SYSTEM ALLOWS
US TO PURSUE THE NEXT QUESTION.
SO WITH THE NEXT QUESTION IS HOW
DOES THE O-GlcNac SHUN
EVENT ON CK 2 ALPHA ON THE
PROTEOME. WE DID
PHOSPHOPROTEOMICS. I WILL SHOW
YOU THE VOLCANO PLOT FOR
PHOSPHOSITE CHANGES IN THE
PRESENCE OF THE NANOBODY OGT
SYSTEM COMPARED TO THE
CATALYTICALLY INACTIVE FORM BUT
WE SEE BECAUSE WE HAVE MORE
GLYCOSYLATION WE ARE SEEING
ELEVATION IN SPECIFIC PHOSPHO
PHOSPHOPROTEINS WHEREAS WITH THE
NANOBODY OGA SYSTEM BECAUSE
O-GlcNac EVENTS ON CASEIN
KINASE 2 ALPHA WERE NOT THAT
HIGH NOW REDUCING IT DIDN'T LEAD
TO SUCH A LARGE PERTURBATION IN
THE PHOSPHOPROTEOME. THIS LED US
TO POSTULATE PHOSPHORYLATION ON
KNOWN CK 2 ALPHA SUBSTRATES HDAC
1 AND 2 THE
O-GglcNaCYLATION EVENT
IS AUGMENTING THE
PHOSPHOPROTEOMIC DATA AND
LEADING TO FOSTERED ACTIVITY.
THIS IS AN EXAMPLE WE ARE USING
THIS SYSTEM IN COLLABORATION
WITH OTHERS INCLUDING JOAN
WALKERS LAB A SPECIALIST IN
DROSOPHILA MODELS OF
NEURODEGENERATION AND
NEUROBIOLOGY, SPECIFICALLY
LOOKING AT HOW THE SYSTEM CAN
REGULATE SLEEP AND CIRCADIAN
BEHAVIOR. JIA NIU'S LAB BOSTON
COLLEGE, WE HAVE BEEN INTEGRATE
OUR TOO MANY SYSTEM WITH HIS TO
UNDERSTAND O-GlcNac IN THE
CONTEXT OF TRANSCRIPTIONAL
ACTIVATION AND EMILYN ALEJANDRO
WE MET THROUGH ONE OF THE NIH
MEETINGS SPECIFICALLY ON
O-GlcNac WHO HAS BEEN
PIONEERING USING THE TOOLS FOR
STUDYING DIABETES. AND THE
O-GlcNac IN O-GlcNac
TRANSFERASE IN BETA CELLS SO WE
USE THESE IN COLLABORATION OR
INDEPENDENT OF IT AND WE HAVE
PUT TOOLS IN AUTHORITY, IT'S THE
ONLY TOOLS WE HAVE ON ADD GENE
SO YOU CAN'T MISS THEM IF YOU
LOOK FOR THEM AND THESE TOOLS
HAVE BEEN DISTRIBUTED TO OF
WHICH OVER 50 SAMPLES HAVE BEEN
DISTRIBUTED. WITH THAT, I HOPE I
HAVE ABLE TO SHARE WITH YOU A
LITTLE BIT ABOUT WHAT WE HAVE
DONE OVER THE LAST FEW YEARS IN
THE -- AS PARTS OF THE COMMON
FUND, BOTH IN DEVELOPING THE
TOOLS AND NAVIGATING TO APPLY
THEM AND GOING TOWARDS THE
FUTURE WE ARE EXCITED TO BE ABLE
TO USE THESE AS WELL AS TO
CONTINUE TO DEVELOP NEW
VERSIONS. LAST BUT NOT LEAST I
HAVE TO THANK MY FANTASTIC LAB,
I HAVE HIGHLIGHTED MOST KEY
PLAYERS, AND THE PROJECTED AS I
HAVE GONE AND THANK YOU, VERY
MUCH.
[APPLAUSE]
>> OUR FINAL SPEAKER IN THE
O-GlcNac SESSION IS DR.
AI, UNIVERSITY OF VIRGINIA.
>> CAN YOU HEAR ME? OKAY. YES
IT'S WORKING. I JUST WANT TO
START MY PRESENTATION BY
THANKING PAM AND CAROL AND
CATHERINE FOR ORGANIZING THIS
WHOLE PROGRAM AND MY LAB
ACTUALLY WORKS WITH BIOSCIENCES
TO STUDY NUCLEOTIDE SUGARS. MY
RESEARCH STARTED WITH THIS
PARTICULAR IMPORTANT PROTEIN
GFP. THIS PROTEIN WAS DISCOVERED
IN LATE 1950s, MANY, MANY
YEARS AGO FROM JELLY FISH, FROM
THIS ORGANISM. AND JELLY FISH
HAS USED THIS PROTEIN FOR A
ENERGY TRANSFER SYSTEM FROM A
BIOLUMINESCENT PROTEIN AND THIS
PROTEIN HAS BEEN DEVELOPED AS A
MAJOR MARKER AND IN MY RESEARCH
WE USE AS A SCAFFOLD TO DEVELOP
BIORESEARCH. THE TOOLS IN
RESEARCH I WANT TO REMIND YOU
ABOUT CHEMISTRY, SO THIS
PARTICULAR PROTEIN IS INTERESTED
IN YOU PUT A GENE IN THIS
PROTEIN INTO LIVING CELLS,
LIVING ORGANISMS, SEVERAL
TYROSINE AND SEVERAL TYROSINE,
SORRY, SERINE TYROSINE IN THIS
PARTICULAR PROTEIN CAN UNDERGO
THIS SPONTANEOUS CHEMICAL
REACTION WHICH IS RESPONSIBLE
FOR THIS PARTICULAR PROTEIN,
LOOK AT STRUCTURE IN THE MIDDLE
OF THIS PROTEIN. THE IN MY
RESEARCH WE TRY TO USE PROTEIN
AS A MODULE, AS A SCAFFOLD TO
BRIDGE WITH SOME OTHER ELEMENT
WHICH CAN LEAD TO TARGET IN THIS
PARTICULAR PROJECT TO UDP SUGARS
SO WE HOPE THAT WE CAN BUILD A
HYBRID MOLECULE AND BIND WITH
THIS BINDING MODULE CAN MODULATE
FLUORESCENCE OF GREEN
FLUORESCENT PROTEIN. FOR THE
FIRST PART I WILL TALK ABOUT A
SPACE WHICH CAN SENSE UDP
GLCNAC. SO FOR THIS PARTICULAR
AUDIENCE I DON'T IMMEDIATE TO GO
THROUGH WHY THIS MOLECULE IS
IMPORTANT, BASICALLY IT IS A
MOLECULE WHICH BRIDGE METABOLITE
NUTRITION WITH SIGNAL DISEASE,
MY RESEARCH LOOK AT UDP GLCNAC
THIS PARTICULAR MOLECULE, WHICH
DISCUSSED IN THE PREVIOUS THREE
LECTURES, THE NEXT STEP TO
ENZYMES TO USE THIS MOLECULE AS
A SUBSTRATE TO MODIFY PROTEINS.
THEREFORE WE ARE STUDYING THIS
PARTICULAR SMALL MOLECULE, IN
LIVING CELLS AND INDICATOR WE
STUDY THIS BACTERIA UDP GLCNAC
TRANSFERASE AND THIS IS ENZYME
BASICALLY USED TO MODIFY LIPID
MOLECULE IN BACTERIA SO OUR HOPE
IS TO TRY TO GENERATE A HYBRID
MOLECULE WITH THIS GREEN
FLUORESCENT PROTEIN AND THIS UDP
GLCNAC BINDING PROTEIN AND WE
DID HOPE ACTUALLY TO GERMINATE
HYBRID MOLECULE TO CHANGE
FLUORESCENCE ON BINDING WITH
GLCNAC. I DON'T HAVE TIME TO
TELL DETAILS BUT BASICALLY
THROUGH COMPUTATIONAL ANALYSIS
AND EVOLUTION, WE EVENTUALLY
ENGINEER PROTEIN CALLED UGAG
WHICH CHANGE FLUORESCENCE BY
SEVEN FOLD IN RESPONSE TO UDP
GLCNAC. BASICALLY THE MIDDLE
SHOWS THE FLUORESCENCE
EXCITATION AND EMISSION OF THIS
PROTEIN AFTER BINDING WITH
GLCNAC, CHANGE CITATION WHICH
METRIC RESPONSE. THIS PROTEIN
SHOWED WHERE HIGH SPECIFICITY IN
RESPONSE TO UDP SUGARS FOR
EXAMPLE UDP GLCNAC HAS ONE
POSITION CHEMISTRY DIFFERENCE
BUT IT CAN DISTINGUISH GLCNAC
WITH GALNAC. YES DID NOR ASSAY
WE FIND -- MORE ASSAY WE FIND
THIS PROTEIN PART OF THE UDP.
THEN WE LOOK AT LITERATURE IN
ALL PAPERS FROM 2004 FROM
SUSAN'S WORK AND THIS PROTEIN IS
THE SAME AFFINITY. AND WE TRIED
-- IT WAS PROPOSED AS A
MECHANISM UDP AS A CANDIDATE
CELL REGULATOR OF THIS ENZYME
WHICH CAN (INAUDIBLE) ENZYME
ACTIVITY. WE TRIED HARD TO
ENGINEER ENZYME TO HAVE HIGH
SPECIFICITY DISTINGUISH UDP FROM
UDP GLCNAC. WE HAD SPS ALMOST NO
ACTIVITY WITH UDP GLCNAC BUT HAS
NEGATIVE CONTROL SENSOR. SO FOR
OUR SPECIFICITY, WE HAVE TO USE
THIS TWO SENSES TOGETHER. AND I
FOR THIS PARTICULAR EXPERIMENT
ON THIS SLIDE WE TRIED TO ADD
GLUCOSAMINE INTO THE CELL, SHOWS
THIS UDP GLCNAC SYSTEM PATHWAY
SO YOU ADD GLUCOSAMINE TO THE
CELL YOU CAN SEE IN THE UDP
GLCNAC SYNTHESIS AND AT THE SAME
TIME ADD UDP. GLUCOSAMINE IN THE
UPPER RIGHT CORNER, THE DATA IS
WORKING SO IT IS HARD TO CONTROL
THIS POINT. IN GLUCOSAMINE WE
SEE THIS INCREASE OF UDP GLCNAC
IN LIVING CELLS AND AT THE SAME
TIME WE SAW OBSERVE THE UXPS
DECREASE, BASICALLY THIS
INDICATES UTP CONCENTRATION
DECREASE IN LIVING CELLS BECAUSE
THIS CONSUMING PATHWAY. WE HAD
ANOTHER DATA MUTANT DATA
ESSENTIALLY WHICH SHOWS IN
RESPONSE TO UDP GLCNAC. THE
OTHER EXPERIMENT BY KNOCK DOWN
ENZYMES AND OVEREXPRESS ENZYMES
GFAP AND UAP AS EXPECTED THE
CONSENSUS OVEREXPRESS ENZYME AND
ALSO GENETIC KNOCK DOWN. MAYBE I
SHOULD HAVE USED THIS LASER
POINTER. USE THIS MOUSE I GUESS.
SO THE SAME THING OF THE WORK
ENZYME INHIBITOR LIKE
(INAUDIBLE) RESPONDED TO GFAP
INHIBITION. WE ONCE STUDY THIS
USE THIS TO STUDY UDP GLCNAC
CONCENTRATION IN MIN 6 CELLS.
BETA CELLS, CAN INHIBIT RESPONSE
TO GLUCOSE. SO WE TRY TO STUDY
WHETHER OR NOT UDP GLCNAC
CONCENTRATION IS SENSITIVE THE
GLUCOSE IN BETA CELLS, BASICALLY
SHOWS WE FIRST VALIDATED WASN'T
WORKING BETA CELLS BY LOOKING AT
ADDITIONAL GLUCOSAMINE. SO THEN
WE STARTED WITH GLUCOSE TO OUR
SURPRISE THE GLUCOSE
CONCENTRATION BY 2 MILLIMOLAR TO
25 MILLIMOLAR CONCENTRATION,
DIDN'T CHANGE GLCNAC
CONCENTRATION TOO MUCH. AND IN
ORDER THE STAY LIKE IN THE
GLUCOSE CHAIN MODULATION UDP
GLCNAC WE HAD TO USE ZERO
MILIMISSOURI LAR AND OBSERVE --
MILLIMOLAR AND OBSERVE THE
CHANGE. THIS WAS KIND OF A QUITE
A SURPRISE BUT BASICALLY OUR
STUDY SHOWS BEAUTIFUL GLCNAC
LEVEL CELLS IS -- MIN 6 CELLS IS
NOT SENSITIVE TO GLUCOSE
CONCENTRATION. IT IS QUITE
REGULATED. MEANWHILE WE ALSO
STUDY ENGINEERED FLUORESCENT
ORIGINATING GLCNAC AND THIS WILL
IS ALPHA RESPONSE. UDP GLCNAC
INCREASE, TWO DECREASE, 2
MILLIMOLAR BUT A DIFFERENT
SPECIFICITY, IT SEEMS AT LEAST
FOR THE MOLECULE ONLY UDP GLCNAC
AND GALNAC INDUCE A RESPONSE
HERE. WE WERE ABLE TO LOCALIZE
TO HERE COMPATIBLE WITH MORE UDP
GLCNAC AND OBSERVE 2 DG AND
GLUCOSAMINE INDUCED UDP GLCNAC
CONCENTRATION CHANGE INDUCE
COMPARTMENT WITH HIGH
CONCENTRATION OF UDP GLCNAC. IN
ADDITION TO UDP GLCNAC WE
ENGINEERED A SENSE OF GLUCOSE.
UDP GLUCOSE IS MOLECULE,
IMPORTANT FOR GLUCOSE
CONCENTRATION REGULATION IN OUR
BODY. ENERGY STORAGE. SO THAT IS
UDP GLUCOSE IS MOLECULE
INTERMEDIATE TO STORE GROUP TO
UNIFORM GLYCOGEN. THIS IS VERY
IMPORTANT FOR GLUCOSE
CONCENTRATION REGULATION IN
BLOOD. WE INHIBITOR OF GFP TO
PROTEIN WE CALL UGP, IT IS UDP
GLUCOSE PIE ROW PHOSPHORYLATION.
SO ENZYMES HAVE O NO CATALYTIC
ACTIVITY AND WE MADE THIS IN THE
MOLECULE RESPONDING TO UDP
GLUCOSE. WE STARTED WITH
MODULATION WITH K ENZYMES
BASICALLY KNOCK DOWN PGM AND
KNOCK DOWN UGP AND WE CAN
OBSERVE RESPONSE AND ALSO
OVEREXPRESS CO-ENZYME, OBSERVE
ALSO RESPONSE. WE UNTIL RECENTLY
TRIED TO EXPRESS THE SAME IN A
ZEBRAFISH ORGANISM. WHICH CAN
CONFER EXPLORATION OF PROTEIN IN
MUSCLE CELLS AND WE CAN OBSERVE
SINGLE CELL RESOLUTION WE ADD
MOLECULE FOR EPINEPHRIN MOLECULE
ON THE RIGHT SIDE, THIS CAN
INCREASE CMP CONCENTRATION IN
CELLS AND THIS ACTIVITY --
PROTEIN KINASE A WHICH DO TWO
THINGS WITH THIS UDP GLUCOSE,
ONE IS INCREASE GLYCOGEN
CONVERSION TO GLUCOSE 1
PHOSPHATE. AND THE OTHER ONE IS
INHIBITED CENSUS, THIS MOLECULE
WE ARE INCREASE UDP GLUCOSE
CONCENTRATION, I FORGOT TO
MENTION THIS MOLECULE IS THIS
SENSOR OUR SENSOR IS HAVE
REVERSE RESPONSE SO WHEN GLUCOSE
INCREASE WE OBSERVE THE DECREASE
IN FLUORESCENCE. SO WE OBSERVE
THE MOLECULE IN ZEBRAFISH WE
OBSERVE INCREASE OF UDP GLUCOSE.
ABOUT 30 MINUTES THIS RESPONSE
IS REVERSED SO BASICALLY WE
COULD OBSERVE OF THE
STIMULATION, -- EPINEPHRINE
STIMULATION. WE OBSERVE SIGNAL
DYNAMICS TO INCREASE UDP
GLYCOGEN SYNTHESIS AND INCREASE
WITH RUBBING GLYCOGEN LYSIS AND
IN 30 MINUTES WITH THIS COULD BE
GLUCOSE CITRATEED FROM THE
MUSCLE CELL OUTSIDE OF THE CELL
OR MECHANISM WE DON'T QUITE
UNDERSTAND BUT TERMINATE THIS
SIGNALING EVENT AFTER 30
MINUTES. TO SUMMARIZE WE
ENGINEER GENETIC ENCODED SENSE
IN POLYGLYCOSIDE AND ENGINEERED
GENETICALLY ENCODED UDP GLCNAC,
AND FOLLOWED WITH CELLS
INHIBITORS NOT BOUND TO
OVEREXPRESSION OF ENZYMES AND WE
MONITOR UDP GLCNAC LEVELS IN
PANCREATIC BETA CELLS AND
SUGGEST GLUCOSE METABOLISM HPV
IS TIGHTLY REGULATED IN BETA
CELLS AND EXPRESS UDP GLUCOSE IN
ZEBRAFISH AND OBSERVE UDP
GLUCOSE CONCENTRATION CHANGE IN
RESPONSE TO EPINEPHRINE SO WE
THINK THIS EVENTUALLY HELP US
UNDERSTAND DISEASE SUCH AS
DIABETES FOR EXAMPLE. AND THEN
WE -- OUR RESEARCH IS SUPPORTED
BY GRANTS. THE WORK WE PRESENTED
TODAY WERE DONE MOSTLY BY THESE
POST DOCS ZEFAL LI, WHO IS HERE
AND HAS A POSTER AND YOU CAN ASK
HIM MORE DETAILS, SOMETIMES MORE
DETAILS I DON'T KNOW. AND
ANOTHER POST-DOC JING ZHANG
COMPLETED THIS WORK. IF YOU
DON'T FIND ENERGY SEND ME AN
EMAIL. I'M HAPPY TO SHARE MY
MATERIAL. ADD GENE ABOUT 100,000
SO YEAH. THANK YOU.
[APPLAUSE]
>> THAT CONCLUDES OUR
O-GlcNac SESSION NOW I
HAND IT TO NATASHA FOR
GLYCOPROTEOMICS. THE FIRST
SPEAKER IN THE GLYCOPROTEOMICS
SESSION IS DR. ROBERT WOODS CCRC
UNIVERSITY OF GEORGIA TALKING
ABOUT GLYCAN STRUCTURE PRE-AND
POST DEPOSITION IN THE PDP.
>> LIKE ALL SPEAKERS I'M
GENUINELY WANTING TO THANK THE
NIH PAM, CARL, DOUG, FOR THE
UNFAILING SUPPORT OF THE COMMON
FUND INITIATIVE AND EVEN BEFORE
THAT. AS A TOOL DEVELOPER I CAST
MY MIND BACK TO THE YEARS BEFORE
WE HAD THE TOOL INITIATIVE AND
BOY IT WAS BLEAK AND DIFFICULT
TO GET FUNDED SO THIS WAS
TREMENDOUS FOR US. I WILL TALK
TODAY ABOUT AN INITIATIVE WE HAD
THAT WAS TOGETHER WITH JASMINE
YOUNG, THIS WAS ONE OF THE BIG
PROBLEMS WE WERE AWARE OF IN THE
PDP. WHEN I SAY WE ALL, I MEAN
ALL THE OF US OBSESSED WITH 3-D
STRUCTURE AS CATH RIB SAID. --
CATHERINE SAID. SO TURNS OUT AS
OF MAY -- SO THIS MONTH THERE
ARE ALMOST 190,000 MACRO
MOLECULAR STRUCTURES IN THE PDB.
USING THIS PROGRAM WE KNOW
CARBOHYDRATES ARE AVAILABLE IN
19,000 PDP OR 10% OF THE PDB.
THIS WASN'T KNOWABLE UNTIL WE
DID THIS. IT WAS ESTIMATED
VARIOUS WAYS BUT THE PROBLEM WAS
THAT THE PDB HAD NO STANDARD
NOMENCLATURE AND MANY ERRORS
MANY THE THE NOMENCLATURE SO YOU
COULDN'T SEARCH AND FIND SUGARS.
AND EVEN TODAY, THERE'S SEARCH
INTERFACES IS EXTRAORDINARILY
LIMITED SO WE CREATED OUR OWN
INTERFACE FOR RESEARCH IN PDB.
BECAUSE OF LACK OF ATTENTION TO
CARBOHYDRATES EARLY ON IN THE
PDB DEPOSITION THERE ARE LOTS OF
INCONSISTENCIES THAT PUSH THE
PDB TO WORK TOGETHER WITH US TO
DO WHAT THEY CALL REMEDIATION.
WHICH IS GOING BACK THROUGH THE
DATA, FINDING ERRORS IN
STRUCTURES WHICH THEY DON'T
CORRECT BUT FLAG AND ERROR MS.
NOMENCLATURE AND SO FORTH. THIS
-- UNTIL THEY DID THIS THIS
CAUSED A LOT OF CONFUSION.
BECAUSE WHEN STRUCTURE IS
DEPOSITED PEOPLE LIKE ME WHO DO
MODELING AND RETRIEVE THE
STRUCTURE THINK IT IS RIGHT
BECAUSE IT WAS PUT THERE. AND I
TELL YOU WHAT, IF WE ARE
MODELING PEOPLE TRYING TO
CONVINCE PEOPLE WE ARE WRITING
CRYSTAL STRUCTURE DRAWING THAT
IS HARD BATTLE SO THIS IS A WAY
TO OBJECTIVELY LOOK AT THAT AND
FIGURE WHAT IS RIGHT AND WRONG
AND HELPS WHEN YOU ARE TRYING TO
DO THINGS LIKE MOLECULAR
MODELING OR PREDICTION OF
STRUCTURE OR THAT SORT OF THING.
THERE'S TENS OF THOUSANDS OF
COORDINATE SETS OF SUGARS IN THE
PDB THAT'S HUNDREDS OF THOUSANDS
OF HOURS OF CRYSTALLOGRAPHERS
TIME WHEN IT WASN'T AUTOMATED
SOLUTION OF CRYSTAL STRUCTURE,
THIS IS MANUAL WORK TO GET THESE
IN THERE THERE. AND U YOU COULDN'T
FIND THEM, IT WAS REALLY A
PROBLEM. SO PDB WENT FORWARD
WITH REMEDIATION, TOOK WHAT WE
DEVELOPED AND USED IT TO FIND
MISTAKES AND WENT THROUGH AND
14,000 FILES WERE REMEDIATED.
IF I CAN GO BACK. 18,000 EXIST.
SO 14 OUT OF 18 HAD PROBLEMS.
THAT'S A THING TO THINK ABOUT.
COUPLE OF THINGS I HEME HAPPY
WITH THEY SCRIBED
OLIGOSACCHARIDES WITH POLYMERS
OF MONOSACCHARIDES. IT WASN'T
THAT WAY INITIALLY, THEY WERE
JUST CRYSTALLOGRAPHER WE CALLED
THEM MAN 3. IT WAS MAN ALPHA 1,
2, BLAH BLAH, JUST CALL IT, IT
IS A LIGAND. THEY INTRODUCED
3-DS AND FGG. THIS CAME OUT OF
THIS WORK. YOU CAN SEE HERE THE
STANDARD 2 DS FG RECOMMENDATION
AND THEN TO THE RIGHT OF IT THE
UPPER RIGHT CORNER, THE
TRADITIONAL 3-D, PROFOUND
DIFFERENCES. UNLESS YOU ARE
OBSESSED WITH 3-D STRUCTURE
CARBOHYDRATES YOU CANNOT LOOK AT
THE 3-D STRUCTURE UPPER RIGHT
AND SEE WHERE FUCOSE IS. WE
THOUGHT THERE MUST BE A BETTER
WAY SO WE TOOK 2 DS AND FG AND
PUT IN AS 3-D ICONS OR 3-D
SYMBOLS. AND I THINK THIS IS AS
REVOLUTIONARY AS USING RIBBON
STRUCTURE FOR PROTEINS. IT HELPS
YOU IMMEDIATELY SEE THE KEY
THINGS GOING ON AND ONE OF THE
THANKS POINTS OUT IS THIS
WONDERFUL LITTLE LESS THAN FG
NOTATION THAT IS SYMMETRIC IS
NOT REPRESENTATIVE OF MOLECULAR
STRUCTURE. THESE ARE NOT
SYMMETRIC MOLECULES. SO ONE OF
THE TOOLS WE DEVELOPED IS THIS
SEARCHING FOR GLYCANS IN THE PDB
SO GO O THIS WEB SITES GLIA
CONDITION.ORG YOU CAN DO THIS.
AS LARRY TABAK SAID ON THE FIRST
DAY, WE ARE SUPPOSED TO BE
DEVELOPING TOOLS FOR
NON-SPECIALISTS. AND THAT IS
EXACTLY THE WAY WE DID THIS. WE
TOOK OUR OLD WEBSITE AND SAID
NO, NO, THAT IS FOR SPECIALISTS
WHAT COULD GYCO DO? NOBODY LIKES
GETTING INSURANCE, SO YOU GO TO
WEBSITE SEEMS EASY, WE THOUGHT
MUST BE EQUIVALENT THING FOR US
SO WE TRIED TO KEEP THAT MIND
SET OF HOW TO DO.
THIS AND WE DECIDED BY
INTERROGATION BASED BUTTONS SO
WHEN A NON-SPECIALIST LANDS
THERE AND SAYS WHAT ARE THE
SHAPE OF MY GLYCANS? THAT IS
WHAT I WANT TO KNOW. SO IT IS
MORE OBVIOUS THAN HOW IT WAS
BEFORE. WHEN YOU DO THIS, THERE
IS MULTIPLE WAYS TO FIND GLYCANS
IN THE PDB NOW. WE ARE GOING TO
-- I WILL SHOW YOU A POINT AND
CLICK I WISH I COULD DO IT LIVE
BUT WE WILL DO IT THIS WAY. BUT
GO TO GLYCAN.ORG DOES HE
UNDERSTAND THIS FOR YOURSELF. IT
WILL POP UP THIS POINT AND CLICK
SCREEN. NOTICE A COUPLE OF
THINGS, ONE YOU SEE THE LITTLE
STAR, WILD CARD, THAT IS TO HELP
YOU SEARCH, ANYTHING IN FRONT
CAN BE WHATEVER YOU WANT JUST
LIKE YOU THINK A WILD CARD DOES.
SO THIS SEARCH FINDS ANYTHING
WITH -- NS TO GLUCOSAMINE IN
PYRANOSE FORM. IT IS FAST AND
THE REASON IT IS FAST IS WE
WROTE A NEW PDB. WE CREATED A
DATABASE THAT IS CUSTOMIZED FOR
STORING DATA FOR SUGARS THAT WE
PULL OUT OF THE PDB. WE STORE
ALL THE DATA IN ANY ENTRY THAT
HAS A SUGAR. THAT MEANS WE CAN
CREATE OUR OWN WAY TO STORE THE
DATA IN OUR OWN WAY TO SEARCH
THE DATA. WE UPDATE THIS
PERIODICALLY SO IT IS ALWAYS UP
TO DATE. WHEN YOU CLICK ON IT
RIGHT AWAY, IT SEARCHES 95,000
ENTRIES WITH SUGARS. IT
PROVIDES BACK UNIFORM
NOMENCLATURE WHAT THE SUGAR IS
IN THE STRUCTURE IT FOUND.
PROVIDES LINK TO PDB ENTRY, A
LINK TO THE VIEWER OF THE
STRUCTURE IF YOU WANT TO SPIN IT
AROUND AND DROP DOWN TO DISPLAY
MORE INFO. WHEN I TALK UNIFORM
NOMENCLATURE LET'S LOOK AT THESE
EXAMPLES. LOOK AT THE ENDS OF
THEM, ONE IS ATTACHED TO A
GLUTENATE. SUGAR ATTACHED TO
GLUTAMATE, NOT SUPER COMMON BUT
THERE IT IS. THERE IS A SUGAR
WITH OH ON THE END. THAT IS NOT
ATTACHED TO ANYTHING. CLEARLY
THAT IS A LIGANDS. AND THERE IS
SUGAR ATTACHED TO ASPEAR GENE SO
THAT IS A IMMUNOGLYCAN.
SO THIS REPRESENTS THE THREE
TYPES OF THINGS TO PULL OUT OF
THE DATA. IF YOU CLICK MORE
BUTTON YOU GET A COUPLE OF BITS
OF INFORMATION THAT I THINK ARE
QUITE USEFUL. IN TERMS OF THE
QUALITY OF STRUCTURE,
CRYSTALLOGRAPHERS OFTEN REFER TO
B FACTOR WHICH IS THE
UNCERTAINTY OF THE ATOMIC
POSITION AND THEIR STRUCTURE.
AND THEY WILL REPORT SOMETIMES
THE MEAN B FACTOR OF THE PROTEIN
BUT NOT OF ANYTHING ELSE.
BECAUSE THESE SUGARS ARE POORLY
RESOLVED WE THOUGHT LET'S REPORT
THE MEAN B FACTOR OF THE
OLIGOSACCHARIDE. SO NOW WHEN
YOU DO THIS, AND THINK IS THIS
STRUCTURE REASONABLE, THIS CAME
OUT RECENT WILL I IN A
PUBLICATION COMING OUT WITH BOB
WHERE E H SAID FUCOSE IS IN A
DIFFERENT ORIENTATION THAN YOUR
MODEL SAYS, THE MEAN B FACTOR IS
SOMETHING 65 OVER 95. IT IS
FANTASY LAND. KNOWING THIS YOU
CAN HAVE QUANTITATIVE ASSESSMENT
OF THE QUALITY OF THE
COORDINATES FOR THE SUGAR. THE
OTHER THING YOU GET IS SOMETHING
ON THE BOTTOM, IF THERE IS
ANYTHING THAT DISAGREES BETWEEN
PDB NOMENCLATURE OR DATA, AND
WHAT WE THINK IT IS. IN THIS
CASE IF YOU LOOK AT THE
STRUCTURE IT IS MURNAC.
CURRENTLY IN OUR NAMING SYSTEM
WE DON'T HAVE ONE SO WE CALL
THIS A WEIRD GLCNAC WITH AN RO 3
MODIFICATION. SO THIS IS A
MISMATCH BUT GIVES YOU HIGHLIGHT
SOMETIMES IT IS US THAT DOESN'T
UNDERSTAND, SOMETIMES THE PDB
THAT HAS INCORRECT. IF YOU THEN
CLICK THIS AND WISH I COULD LYE,
IF YOU CAN SEE ICON
REPRESENTATION OF THE SUGAR AND
YOU CAN SPIN AROUND AND LOOK
YOURSELF AT THE 3-D STRUCTURE.
IN TERMS OF PROBETY WE CALLED
THE TOOLS GLY FINDER AND
GLYPROBITY. THEY MAY SPLIT OUT,
GLYPROBITY IS NAMED AFTER A TOOL
THAT CRYSTALLOGRAPHERS USE FOR
LOOKING AT CONFIDENCE OF THE
COORDINATES OF THE PROTEIN.
THERE IS SOME NEED FOR THIS FOR
SUGARS AS WELL. ONE THING WE
DEVELOPED THAT CAME OUT THIS,
BECAUSE WE DO THEORETICAL MODELK
OF MOLECULAR STRUCTURE, WE ARE
OFTEN LOOKING AT ENERGY OF
DIFFERENT SHAPES. CARBOHYDRATES
HAVE WELL DEFINED CONFIRMATIONAL
PREFERENCE AROUND GLYCOSIDIC
LINKAGE, WE CAN BUILD 3-D MODEL
OF SUGARS BECAUSE WE KNOW HOW
THE LINKAGES BEHAVE, YOU CAN'T
DO IT WITH PROTEINS BECAUSE THEY
ARE TOO FLEXIBLE. ALPHA FOLD
SOLVE PROTEIN MODELING PROBLEM,
THIS RECENTLY, CARBOHYDRATE
MODELING PROBLEM SOLVED FOR A
LONG TIME BECAUSE WE UNDERSTAND
LINKAGES BEHAVE. WHAT THAT LETS
US DO IS CREATE IMAGE LIKE THIS
AKIN TO A RAMISHANDRIN PLOT FOR
PROTEIN AND BLACK DOTS WHERE
WHERE THE CRYSTAL STRUCTURES
HAVE THE THIAN ANGLES, THE
SHAPES. COLORS WE INDEPENDENTLY
PREDICT ENERGY OF THESE
STRUCTURE TO BE. SO YOU CAN SEE
THIS IS A VERY INTERESTING BASED
ON QUANTUM CALCULATION OF ENERGY
SURFACE, AND WHAT IT SHOWS IS
THERE IS -- THE BLUE IS STABLE
STUFF AND THAT IS WHERE MOST
STRUCTURE ARE, IT PROVIDES VERY
OBJECTIVE INDEPENDENT
ASSESSMENT, IT ALSO LETS YOU
FIND WHAT ARE CALLED OUTLIERS.
THIS IS AN INTERESTING TERM,
DOESN 'T MEAN MISTAKE. IT MEANS
STATISTICALLY NOT COMMON. THIS
COULD BE MISTAKE AND PROBABLY IS
WITH SUPER HIGH ENERGY, PROBABLY
INCORRECT STRUCTURE, YOU CAN
FLIP A SUGAR 180 DEGREES IN
ELECTRON DENSITY AND NOT KNOW
THE DIFFERENCE UNLESS YOU ARE
CAREFUL. BUT COULD BE ENZYME HAS
DISTORTED IT. UNLESS YOU FIND
THINGS LIKE THIS. WHAT WE ARE
STRUGGLING WITH, EVOLVING TO DO,
WHEN IS A CARBOHYDRATE NOT A
CARBOHYDRATE? THERE ARE LOTS OF
MODIFICATIONS AND DERIVATIVES.
SO THESE ARE THE THINGS WHERE AT
MOMENT WE CALL THEM R GROUPS AND
WE WILL LIST THE C 612OH OF THE
R GROWN BUT GIVE IT A NAME
BECAUSE IT IS JUST NOT WORTH THE
EFFORT OF TRYING TO NAME THESE R
GROUPS. BUT IT DOES DETECT THEM
AND THIS IS A VERY POWERFUL
THING THAT WHEN WE FIRST STARTED
TO THIS PROJECT WE WEREN'T
THINKING THIS, WE WERE THINKING
OF LOOKING FOR CAR BOAR HYDRATES
BUT WE GOT SUPPLEMENT THAT
HELPED US GET THE MODIFICATIONS
AS WELL. WHAT DID WE LEARN FROM
THIS? JUST LOOKING THERE IS 54,
55% SUGARS IN THE PDB ARE N
LINKED SUGARS. SO SUGARS FAR
FEWER. I DON'T KNOW IF THAT IS
BECAUSE THEY ARE NOT LOOKED FOR,
WHICH IS DEFINITELY POSSIBLE. OR
JUST NOT THERE. CEILINGS ARE
FURTHER -- C LUNGE IS FURTHER
UNLIKELY. WHAT CAN YOU DO A
LITTLE FACTOID TO AMUSE FRIENDS,
52,000 IMMUNOGLYCANS OUT OF
7,000 SOME ODD STRUCTURE
CONTAINING N LINK GLYCANS THAT
MEANS SOME N LINK GLYCANS ON
AVERAGE PER GLYCOPROTEIN. WHO
KNEW THIS? NOBODY. WE COULDN'T.
DOES IT MATTER I DON'T KNOW BUT
IT IS A FACT. THAT IS ALL I WANT
TO TALK ABOUT. I WILL THANK THE
PEOPLE INVOLVED. THIS IS THE GLY
FINDER WORK UNDERTALKEN BY A
TALENTED GRAD STUDENT DAVID
MONTGOMERY. I WOULD LIKE TO
ACKNOWLEDGE THE NIH AND
UNIVERSITY OF GEORGIA AND ALL
THE TEAM, YOU GUYS FOR THE TIME.
THANK YOU.
>> NEXT SPEAKER IS DR. RILEY AND
HE TALK ABOUT MAKING
GLYCOPROTEOMICS MORE ACCESSIBLE.
>> THANK YOU, MICROPHONE WORKS
TOO. THANK YOU ALL FOR ALLOWING
ME TO GIVE THIS PRESENTATION,
HERE REPRESENTING BERTOZZI GROUP
AT STANFORD, THANK YOU FOR
SENDING ME TO PRESENT TO Y'ALL
AND TO ORGANIZE ORS FOR AGREEING
TO LET ME DO THIS. I'M
PRESENTING ON THIS UO 1 SHARED
BETWEEN CAROLYN AND SHARON AT
STANFORD. AND WE ARE EXCITED TO
TALK ABOUT BOTH WHAT WE HAVE
DONE IN THE FUNDING PERIOD AND
ALSO WHAT THIS WORK IS GENERATED
AS YOUR NEXT IDEAS AND WORKING
ON MOST RECENT FEW YEARS.
CONFLICT OF INTEREST, CAROLYN
HAS A FEW COMPANIES SHE'S PART
OF SO MENTION THAT. I HOPE MY
STORY IS ONE SMALL
REPRESENTATION OF SUCCESS OF
WHAT THE GLYCOSCIENCE COMMON
FUND HAS BEEN. THAT'S MY THIRD
IN PERSON COMMON FUND I WENT TO
BECAUSE IN GRADUATE SCHOOL AS
MEMBER PROFESSOR JOSH COUNSEL'S
GROUP UW MADISON MADISON. WE GOT AN R21
TO WORK ON HARDWARE DEVELOPMENT
HOW TO ANALYZE GLYCOPEPTIDES
USING INNOVATIVE ELECTRON BASED
AUGMENTATION WE WORKED ON IN
MASS SPEC. IN THOSE MEETINGS,
FIRST TIME I WAS IN THE ROOM
WITH CAROLYN SHE ASKED QUESTION
ABOUT PRESENTATION AND LITTLE
DID I KNOW IN 2016 THAT HAPPENED
AND CONTACT OR TWO LATER DO YOU
HAVE POST DOC OPENINGS, HERE I
AM SIX YEARS AFTER FACT. IT IS
EXCITING TO TALK ABOUT THE UO 1
WITH HER. THIS FUNDING PERIOD
ENDED A FEW YEARS AGO. WHAT IS
INTERESTING ABOUT THIS IS WORK
THAT CAME OUT OF UO 1 GRANT WAS
LOTS OF SPEARHEADED BY KRISTINA
WOO WHO TALK ABOUT HER WORK IN
FRONT OF YOU AND YOU. WE WANT TO
HIGHLIGHT HOW THAT TRANSLATED TO
PRODUCT AVAILABLE IN COMMUNITY
AND WAYS WE ARE STILL TRYING TO
MAKE GLYCOPROTEOMICS AND MASS
SPECTROMETRY ACCESSIBLE TO THOSE
WITH GLYCOPROTEINS. AS WE TALKED
ABOUT DIVERSE GLYCOBIOLOGY
DIVERSE SETS OF TOOLS AND
CAROLYN APPROACH TO THIS IS TO
USE THESE METS BOLLICLY
INCORPORATED SUGARS AND
NUCLEOTIDE SUGARS THROUGHOUT
THESE PRESENTATIONS AND
INCORPORATE INTO GLYCAN TO DO
INTERESTING COOL CHEMISTRY.
GLYCOTON JEW GATES. YOU CAN
TARGET GLYCANS DEPENDING ON TYPE
OF METABOLIC SUGAR YOU
INCORPORATE THAT. WITH Z FOCUS
ON THIS CYTOTAG PLATFORM TO USE
ANY SUGAR YOU CAN IMAGINE TO
HAVE METABOLIC REPORTER THAT IS
FUNCTIONALLIZE.MENT SO YOU CAN
INCORPORATE THIS SUGAR INTO THE
GLYCOCALIX OR INTO
O-GlcNac PROTEINS AS WELL
DEPENDING WHICH MONOSACCHARIDES
YOU USE AND NEEDS TO BE
PRESENTED ON CELL SURFACE OR
O-GlcNacATEED
INTRACELLULAR PROTEINS TOO.
THIS IS A HANDLE ON
GLYCOCONJUGATES AS WE HAVE SEEN
THROUGHOUT PRESENTATIONS TODAY.
WITH ISOTYPE PROTEIN ONCE YOU
HAVE THE LABELED PROTEIN, THIS
GLYCOCONJUGATE HOW YOU CAPTURE
AND DO MASS SPEC TO UNDERSTAND
NOT THAT IT WAS GLYCOSYLATED BUT
WHAT IS THE GLYCOSIDE AND WHAT
GLYCAN WAS THERE SO LOOKING AT
INTACT GLYCOPEPTIDES. AND THEN
IF YOU HAVE A CHEMICAL
REPRESENTATION HOW THIS MIGHT
WORK YOU CAN DEVELOP REAGENT,
THIS IS ISOTAG REAGENT BY
KRISTINA AND THE BERTOZZI LAB,
TO BE ABLE TO CLICK THIS ON WITH
CHEMISTRY TO OUR LABEL
GLYCOPROTEIN DO ON B DIGESTION
SOME WAY TO PURIFY LABELED
GLYCOPROTEINS AND LINK THROUGH
ASSAY LINKERS, DIFFERENT WAYS TO
RELEASE GLYCOCONJUGATE TO BE
ABLE TO ANALYZE. SO WHAT THESE
TAGS LOOK LIKE THESE THINGS HAVE
MULTI-FUNCTIONAL PARTS TO THEM.
WE HAVE THE BIOTIN GROUP HERE
USED FOR BASED ENRICHMENT, THE
LINKER THAT GETS TO CLEAVAGE
SIDE WHICH IS WHERE YOU RELEASE
THIS MOLECULE FROM ENRICHMENT
AND YOU HAVE ISOTOPE RECODING OR
RECORDING. AND SO HERE YOU
INTRODUCE AN ISOTOPIC PATTERN TO
DIFFERENCE FROM PATTERN PRESENT
IN GLYCOPROTENSE OR GLYCOPEP
SIDES, THIS IN YOUR MASS
SPECTROMETER IS A DIFFERENT
PATTERN. THIS IS A NOW ENRICHED
GLYCOCONJUGATE LIKE A PEPTIDE
PROTEIN OF SYSTEM BILL THAT I
WANT TO STUDY. WITH CLICKABLE
SIDE YOU INTRODUCE CHEMICAL
FUNCTIONALITIES TO ATTACH TO THE
METABOLIC SUGAR YOU HAVE
INCORPORATE WITH THE RECIPROCAL
FUNCTIONALITY ON I ALABAMATY ON IT. SO
THIS IN-- ISOTAG INCORPORATES
THE IDEA, THE SECOND PART IS HOW
THE ANALYZE WITH MASS
SPECTROMETRY, THAT WAS THE IDEA
OF UO 1 TO BRING THIS IDEA TO
THE MASSES SO PEOPLE CAN DO
GLYCOPEPTIDE MEASUREMENTS. SO
THE IDEA HERE WITH THE ISOTOPE
RECODING IS YOU HAVE THIS IDEA
THAT WE NOW HAVE A ISOTOPE THAT
LOOKS DIFFERENCE THAN NORMAL
PEPTIDE WOULD. WE CAN TELL MASS
SPECTROMETER GO AFTER THAT AND
CLEAVE -- ANALYZE THAT OVER
PREFERENCE OF ANYTHING ELSE AND
THAT GIVES YOU DATA THAT CAN SAY
THESE SPECTRA SHOULD BE
GLYCOPEPTIDES. SO WITH THIS
IDEA WE CAN ANALYZE SPECIALIZED
SOFTWARE, WORK WITH PROTEIN
METRICS TO -- JENNIFER KOHLER
MENTIONED WORKING WITH THEM AS
PART OF FUNDING MECHANISM, SAME
WITH BERTOZZI LAB TO BE ABLE TO
CHARACTERIZE SPECIFIC
GLYCOPEPTIDES THAT WERE ENRICHED
THROUGH THE ISOTAG PLATFORM. I
WILL MENTION TOO THE GOAL OF THE
UO 1 WAS TO GENERATE A
COMMERCIALLY AVAILABLE PRODUCT
PEOPLE CAN DO THIS SO THIS HAS
COME TO FRUITION THROUGH CLICK
CHEMISTRY TOOLS, YOU CAN SEE
THERE IS MULTIPLE OPTIONS YOU
CAN PURCHASE AND DO THIS YOUR
LAB FOR RELATIVELY AFFORDABLE
PRICES. THE PRICE OF ANTIBODY
YOU MIGHT NEED TO DO FOR
EXPERIMENT. YOU CAN BUY THE
ENTIRE KIT OR YOU CAN BUY JUST
THE LINKER ENRICHMENT SET THAT
TOOL SO IF YOU HAVE YOUR OWN WAY
TO SYNTHESIZE YOUR NUCLEOTIDE
SUGARS, METS BOLLIC
INCORPORATION MECHANISMS YOU CAN
DO THAT AND STILL USE THIS IDEA
OF THE ISOTAG PLATFORM SO YOU
CAN SEE ALL THIS AVAILABLE AT
CLICK CHEMISTRY TOOLS, THIS
REALLY WAS ENTIRE PATH OF THIS
PROJECT US TO BRING TO COMMUNITY
AND NOW EXCITED THAT IT IS
AVAILABLE THAT WAY. KAREN AND
SHARON WERE PIs, KRISTINA DID
WORK TO GENERATE THIS AND THESE
ARE SELECTIVE REFERENCE BUS THEY
HAVE AN ENTIRE BODY OF WORK
TALKING ABOUT HOW ISOTAG IS USED
FOR N GLYCANS O GALNAC AND
O-GlcNac GLYCOPROTENSE SO
DIFFERENT WAYS TO USE THE
TECHNOLOGY AND WE CAN SPEND THE
ENTIRE REST OF THE AFTERNOON
TALKING ABOUT DIFFERENT AWESOME
DATA THEY GENERATED THAT WAY.
BUT INSTEAD WE WANT TO PIVOT,
CAROLYN SAID YOU SHOULD TALK
ABOUT WORK THAT'S GROWN FROM
THIS, WHAT WE ARE DOING NOW WITH
IDEAS FROM THE UO 1 THROUGH THIS
MECHANISM. SO WANTS TO POINT OUT
THIS IDEA OF WHAT IS MS BASED
GLYCOPROTEOMICS AND YOU HAVE
SEEN WORK FLOWS AND WHAT IT
BOILS DOWN TO IS MAIN COMPONENTS
YOU HAVE TO CONSIDER. WE CAN
GIVE SEMESTER LONG COURSES ABOUT
EACH OF THESE TOPICS. ISOTAG
TOUCHED ON SEVERAL POINTS. HOW
DO YOU INCORPORATE HANDLING YOUR
SAMPLES, ENRICH GLYCOPEPTIDES OR
PROTEINS.
HOW YOU ANALYZE WITH MASS
SPECTROMETER AND HOW TO SEARCH
IT. THERE'S PAIN POINTS
ESPECIALLY MORE CHALLENGING
GLYCOCONJUGATES IN FIELD.
SO WE WILL TALK ABOUT DATA
INFORMATICS BUT SAMPLE
PREPARATION CHEMICAL BIOLOGY
APPROACH LOOKING AT FOR SOME
CHALLENGING GLYCOPROTEINS. ONE
OF THE THINGS WE ARE MOST
INTERESTED IN STUDIES OF RECENT
YEARS ARE MUCINS. THESE ARE
MASSIVE OGLYCO PROTEINS WITH
DENSE REGIONS OF GLYCOSYLATION,
MOSTLY STUDIED IN DISEASES LIKE
CANCER, WE THINK OF MUC 1
REGULATED IN EPITHELIAL CANCER,
MUC 16 YOU HEARD ABOUT IN
EARLIER PRESENTATIONS AS MARKER
FOR OVARIAN CANCER. SO IT IS
REALLY INVOLVED IN
IMMUNOREGULATION AND EVASION, A
LOT OF HOST PATHOGEN
INTERACTIONS AND ALSO STRUCTURAL
MECHANOBIOLOGY, NOT JUST
CHEMISTRY OF LIGANDS BUT HOW
THEY REARRANGE CELL SURFACE AN
ALTER WAY SURFACE IS ORGANIZED.
SO LOT OF REASONS TO STUDY
MUCINS BUT THEY FRUSTRATE EVERY
STEP OF WHAT MASS SPEC BASED
GLYCOPROTEOMICS WORK FLOW LOOKS
LIKE SO THIS IS WHERE WE FOCUS
ON EFFORTS IN BERTOZZI LAB HOW
TO ADDRESS CHALLENGES WITH
DIFFERENT TOOLS WE CAN IMAGINE
FOR MUCIN O GLYCOPROTEINS. ONE
THING TAKING OFF GAINING
MOMENTUM AS I ENTERED THE LAB
WAS THE IDEA OF USING O
GLYCOPROTEASE. IN THE BERTUZZI
LAB WE SAY MUCINASES, MUCINASES
ARE SELECTIVE TOWARDS MUCINS IN
GENERAL SO THE IDEA HERE IS THAT
YOU CAN HAVE THIS PROTEASE, THIS
ONE IS FROM E. COLI SO A PROTEIN
PUBLISHED ON SINCE EARLY 2000S
AND WE STARTED EXPLORING WHAT
THAT MIGHT LOOK LIKE AND HOW WE
CAN USE IT AND THE TAKE HOME
MESSAGE FROM THIS PLOT IS TO SAY
HERE ARE RECOMBINANTLY EXPRESSED
MUCINS. YOU CAN SEE IF YOU
TREAT THEM WITH STICKY, THE NAME
OF THIS O GLYCOPROTEASE, YOU GET
CLEAVAGE YOU CAN SAY THIS
DEGRADING IT SOMEHOW. THE
PROTEASE THOUGH DOESN'T CLEAVE
BSA NOR DOES IT CLEAVE FET 1 A O
GLYCOSYLATED PROTEIN. SO A LOT
OF EXPLORE FOR RATION WENT INTO
THIS AND BEFORE I GOT INTO THE
LAB FIGURED OUT STCE IS A
MUCINASE IS SELECTIVE FOR THIS
MOTIF THAT EXISTS WITHIN MUCINS
SO THIS TXT, X CAN BE NUMBER OF OF
AMINO ACIDS WHERE THE 3 ANINE
HAS TO BE GLYCOSYLATE SOD YOU
CAN IMAGINE HOW DENSE THE MUCIN
DOMAINS ARE, THIS WOULD APPEAR A
LOT AND STCE CLEAVE IS HERE
BETWEEN SECOND AND THIRD RESIDUE
OF THE MOTIF IT IS NOT JUST THE
GLYCAN AND NOT JUST ABOUT THE
PEPTIDE. THESE MUCINASES REQUIRE
THE GLYCAN AND PEPTIDE MOTIF IN
COMBINATION. AND WE ARE LEARNING
NOW THIS EXCITEMENT IN THE FIELD
THERE ARE MORE MUCINASES THAN
JUST ONE, THERE IS THE FAMILY OF
O GLYCOPROTEASES THAT CAN CLEAVE
NON-MUCINS AS WELL. SO I WOULD
SAY HERE TOO TO HIGHLIGHT HOW
MUCH WORK IS NOW BEING DONE IN
THE BERTOZZI LAB, STACY AND JUDY
HAVE BEEN SPEARHEADING THIS
BEFORE AND GOT IN THE LAB AND
WORK TO DARK RISE NUMBER OF
GLYCOPROTEASES WITH ALL THESE
CLEAVAGE MOTIFS HERE. WE ARE
ALSO LOOKING AT JUDY
SPEARHEADING HOW TO USE INACTIVE
MUCINASES SO THINGS THAT HAVE
SPECIFICITIES FOR THESE DOMAINS
THAT CAN BE INACTIVATED THE F
THEY DON'T CLEAVE BUT STILL
BIND. WITH THESE YOU CAN
IMAGINE HOW YOU CAN USE AS
IMAGING REAGENT AND THINGS IN
THE THAT CAPACITY ALSO LOOKING
HOW WE CAN USE MUCINASES AS A
THERAPEUTIC MODALITY TOO. SO ONE
THING WE ARE USING INACTIVE
MUCINASES FOR AND I DON'T WANT
TO BELABOR THE POINT HOW CAN WE
DETERMINE WHAT MUCIN DOMAIN
GLYCOPROTEINS EXIST IN A SAMPLE,
HOW CAN WE UNDERSTAND WHAT IS
PRESENT IN ORDER TO CHARACTERIZE
WHAT IS THERE. SO WE ARE USING
INACTIVE MUCINASES TO THEN PULL
OUT MUCIN DOMAIN GLYCOPROTEINS.
SO THIS IS SOMETHING THAT YOU
CAN BASICALLY COUPLE INACTIVE
MUCINASE TO A BEAD AND DO SOME
ENRICHMENT THAT WAY AND THIS
SOMETHING THAT STACY SPEARHEADED
AND I HELPED WITH INFORMATICS ON
IS THE IDEA YOU CAN ENRICH MUCIN
DOMAIN GLYCOPROTEINS AND MUCIN
COMPUTATION TO UNDERSTAND THESE
HAVE MUCIN DOMAINS IN THESE
PARTICULAR SPOTS. CHALLENGE
THOUGH OVER THE LAST TALK ABOUT
BEFORE CIRCLE BACK TO THE ENDS
HERE IS IF YOU WANT TO
CHARACTERIZE THE MUCIN SITES
THAT O-GlcNac GLYCOSIDES
IN THE MUCIN DOMAINS IS
CHALLENGING. SO FOR SAKE OF TIME
I WILL SKIP THROUGH MASS SPEC
DETAILS OF WHAT THE
COMPLICATIONS ARE BUT KNOW THAT
HOW YOU FRAGMENT O GLYCOPEPTIDES
DETERMINES LEVEL OF INFORMATION
ACHIEVEED FROM THEM. THE CURRENT
TOOLS DON'T PROVIDES ACCESS TO
THAT INFORMATION THE WAY WE NEED
FOR O-GlcNac
GLYCOPEPTIDES. THERE'S GREAT
TOOLS FOR N GLYCOPEPTIDES BUT O
GLYCOPEPTIDES ARE MORE
CHALLENGING. SO WE TEAM WITH THE
UNIVERSITY OF WISCONSIN TO MAKE
A NEW SOFTWARE ACCESSIBLE AND
AVAILABLE TO THE COMMUNITY AND I
WILL MENTION HOW IT WORKS. WE
LOOK AT DEVELOP TYPES OF
FRAGMENTATION TO IDENTIFY AND
THEN LOCALIZE GLYCOSIDES ON
GLYCOPEPTIDES THAT ARE COMING
FROM THESE MUCIN DOMAINS. I WILL
MENTION FREE OPEN SOURCE
STICKING WITH CHEM OF UO 1
TRYING TO MAKE THINGS AVAILABLE
AND ACCESSIBLE, HAPPY THIS CAN
BE A TOOL PEOPLE CAN DOWNLOAD
AND START USING IF YOU ARE
INTERESTED. WE WILL GO THROUGH
MENTION IT IS QUICK, IDEA WE CAN
DO THESE SEARCHES AND FRACTIONS
OF A MINUTE. AND THAT WE GET AS
GOOD IDs AND OTHER TOOLS OUT
WILL SO WAY TO EXPEDITE WHERE
YOU CAN DO O GLYCOPROTEOMICS ON
MUCIN DOMAIN GLYCOPROTEINS AND
HELPS IN STUDIES YOU CAN SEE
THAT WE ARE ABLE TO NOW SHOW O
GLYCOPEPTIDES COMING FROM THESE
MUCIN ENRICHMENTS THAT WAY. THE
LAST THING I WILL SAY IS THAT
THERE ARE LOTS MORE TOOLS BEING
DEVELOPED IN O GLYCOPROTEOMICS
SPACE. THERE IS RECENT
EVALUATION THAT CAME OUT BUT
TOOLS USED DAY TO DAY WEREN'T
AVAILABLE WHEN THIS STUDY
STARTED IN 2017. SO NOW A SECOND
PHASE OF THIS STUDY, THAT STACY
FORMER POST DOC IN B HE
CANRTOZZI LAB NOW PROFESSOR AT
YALE AND CO-LEADING AS PART OF
THIS INITIATIVE SO WE SEW SAYS
ACTIVELY RECRUITING DEVELOPERS,
WE STOPPED THE PHASE ON SUNDAY
BUT IF YOU ARE INTERESTED OR
KNOW SOMEONE THAT IS, WE CAN
MAKE EXCEPTIONS. CURRENTLY WE
HAVE 18 TEAMS REG DEERED SO VERY
EXCITED ABOUT THIS AND LOOKING
FORWARD TO THAT. SO DON'T WANT
TO TAKE TOO MUCH TIME
ACKNOWLEDGE A LOT OF GREAT
PEOPLE ESPECIALLY THE UO 1 PHASE
THAT WAS THE TEAM BEFORE ME SO
KRISTINA, SHARON, CAROLYN SO
MANY MOTHER PEOPLE AND IF YOU
HAVE INTEREST IN GLYCOPROTEOMICS
INITIATIVE CONTACT US ASAP
THROUGH EMAIL OR TWITTER AS
WELL. THANK YOU FOR TIME.
[APPLAUSE]
NEXT SPEAKER IS DR. SHAPIRO WHO
WILL TALK ABOUT ST85IA6 ON SOLID
TUMORS.
I WOULD LIKE TO THANK
THE ORGANIZERS TO TALK TO YOU
ABOUT THE WORK WE HAVE DONE AND
FOR COMMON FUND FOR ALLOWING ME,
I GUESS I'M ONE OF THE
NON-SPECIALISTS,S NOT REALLY A
GLYCOBIOLOGIST BUT WE BECAME
INTERESTED IN IT BECAUSE WE
STUDY A SIALIC ACID TRANSFERASE
TRYING TO UNDERSTAND THE ROLE IT
HAS IN INHIBITING IMMUNE
RESPONSE THE TUMORS. SO THE ONE
WE STUDY IS ST85IA6 WHICH IS A
SILO TRANSFER' SIGH LITTLE
TRANSFERASE ON O LINKED
GLYCOPROTEINS. THE WORK WE HAVE
DONE IS PRODUCT OF THE ENZYME
BINDS TO AND INHIBITOR RECEPTOR
LOCATED ON IMMUNE CELLS CALLED
SIGLEC E, ON MACROPHAGES
DENDRITIC CELLS AND NEUTROPHILS
AND FUNCTIONS TO INHIBIT
CELLULAR ACTIVATION. CYCLIC E IN
MICE IS SIMILAR TO SIGLEC 7 AND
9 IN HUMANS. SIGLEC SO WE HAD
KNOWN THAT IF YOU ACTUALLY LOOK
AT SOME OF THE CANCER DATABASES,
THAT ST85IA6 IS OVEREXPRESSED IN
MANY SOME TUMORS AND IN SOME
TUMORS CORRELATES WITH POOR
RESPONSE SO WE WANTED TO
UNDERSTAND WHAT STATUAS DOING.
SO WE GENERATED PAIRED CELL
LINES, SO MC 38 IS A MOUSE COLON
CANCER CELL LINE, B 16 F 10 IS A
MELANOMA CELL LINE. AND WE
EITHER HAD ONES NEITHER CELL
LINES EXPRESSED ANY APPRECIABLE
LEVELS OF F ST85IA6 AND WE
GENERATED STABLE CELL LINES,
WHEN WE PUT ST85IA6 IN WE
DEMONSTRATED THEY MADE LIGANDS
FOR SIGLEC E SO WE USED
RECOMBINANT HOOKED UP TO HUMAN
PORTION OF IGG ONE AND COME BACK
WITH FLUORESCENTLY TAGGED
ANTI-HUMAN IGG 1 BY FLOW
CYTOMETRY TO BE ABLE TO QUANTIFY
THE BINDING. SO USING THESE CELL
LINES WE WERE ABLE TO
DEMONSTRATE BLUE IS GOING TO BE
WITH ST85IA6 RED IS WITHOUT.
THAT IN BOTH MODELS THAT THE
PRESENCE OF ST85IA6 ACCELERATED
TUMOR GROWTH AND DECREASE
SURVIVAL WHEN PUT INTO THE
FLANKS OF MICE. WE WERE ABLE TO
SHOW THIS IS NOT A CELL
INTRINSIC EFFECT ON TUMOR GROWTH
BUT EXTRINSIC EFFECT DUE TO
SIGLEC E EXPRESSION IN MOUSE AND
WE WERE ABLE TO DEMONSTRATE THIS
WORK IN VIVO AS WELL BECAUSE WE
GENERATED MOUSE MODELS WHERE WE
CAN CONTROL EXPRESSION OF
ST85IA6 IN A CRE DEPENDENT DOCKS
RELATABLE WAY SO OVEREXPRESSING
ST85IA6 IN EPITHELIAL CELLS IN
THE GUT DOESN'T EFFECT SURVIVAL
OF THE MOUSE. THIS IS AN
ESTABLISHED COLON CANCER MODEL
USING CRE WHICH EXPRESSES
EPITHELIAL CELLS OF THE SMALL
LARGE INTESTINE ALONG WITH
ACTIVATED KRAS IN A TRUNCATED
APC GENERATES COLON CANCER IN
SIX MONTHS. IF WE PUT THESE
TOGETHER OVEREXPRESS ST85IA6 IN
THIS MODEL WHAT HAPPENS IS THAT
TUMORIGENESIS IS ACCELERATED SO
YOU GET TUMOR DEVELOPMENT IN TWO
MONTHS. SO THE WORK WE DONE
INITIALLY IS AGNOSTIC, IT DOES
NOT IDENTIFY PROTEINS IDENTIFIED
BY ST85IA6 WHICH IS LEADING TO
THIS CHANGE IN THE IMMUNE
RESPONSE BUT WE HAD PAIRED CELL
LINES WITH AND WITHOUT ST85IA6
THAT ALLOW POTENTIALLY GO AFTER
THESE TARGETS. SO WE GOT A
SUPPLEMENT TO OUR NCI RO1 TO
IDENTIFY WHAT SOME OF THESE
TARGETS ARE. AND WE WORKED WITH
MAYANK SARASWAT WITH DR. PANDEY
AT MAYO FOR MASS SPEC TO LOOK AT
DIFFERENTIALLY SIALYLATED
TARGETS BETWEEN OUR ST85IA6
EXPRESSING AND NON-EXPRESSING
CELL LINES. SO THIS IS BASICALLY
WHAT AKILESH'S GROUP IN CELL
LINES WITH PULL DOWNS WITH
LEPTONS AND MASS SPEC. SO TODAY
I WOULD LIKE TO TALK BRIEFLY
ABOUT ONE OF THE TARGETS THAT WE
GOT ON THIS SCREEN. AND SO CD4 4
CAME UP AS A POTENTIAL TARGET
FOR ST85IA6 WHICH WAS
DIFFERENTIALLY SIALYLATED WHEN
ST85IA6 WAS EXPRESSED. SO IN
TERMS OF GLYCANS WHICH LOOK TO
BE MODIFIED BY ST85IA6 THEY
APPEAR CORE 1 AND CORE 2 O
GLYCANS BUT THIS DOESN'T
ACTUALLY TELL US WHETHER OR NOT
THESE GLYCANS ON CD4 4 ARE
GENERATING LIGANDS FOR THE
SIGLECS WE ARE BIOLOGICALLY
INTERESTED IN SO TO DO THAT I
DON'T KNOW HOW TO GO BACK. WE
USED HEAP 293 CELLS IN PART
BECAUSE THEY DON'T EXPRESS CD4 4
SO LOOK SPECIFICALLY FOR
CONTRIBUTION OF CD4 4 TO
RECOMBINANT SIGLEC BINDING,
THESE ARE RECOMBINANT CELLS
LINES SO NOT RECOMBINANT E BUT
SIGLEC 7 AND 9 AND DO
CO-TRANSFECTIONS WITH SIGH
CLINICAL TRANSFERASIST SO WE CAN
MARK THE CELLS TRANSFECTED AND
WITH BOTH OF THEM TOGETHER. WE
USE FLOW CYTOMETRY FOR THOSE --
I DON'T KNOW WHY IT IS GOING
AUTOMATICALLY. SO FOR THOSE THAT
-- THIS IS GOING TO FRUSTRATE
ME. I DON'T KNOW WHY IT IS
AUTOMATICALLY ADVANCING. SO IF
WE LOOK AT THE FLOW SIGH TOM TRY
WE WILL DO IT THIS WAY. I HAVE A
DIFFERENT WAY. IF WE LOOK WITH
JUST CD4 TRANSFECTED OR CD4 4
PLUS ST85IA6. WHAT YOU SEE ON
THE X AXIS IS ACTUALLY LOOKING
FOR FLAG TAG CD4 4. SO CELLS
TRANSFECTED WILL BE BOXED ON THE
RIGHT, NON-TRANSFECTED CELLS
WITHIN THIS GROUP ARE ON THE
LEFT. AND WE ARE USING
RECOMBINANT SIC SIGLEC 7 ON Y AXIS
IN THE UP PER FIELD SO IF WE
OVEREXPRESS CD4 4 AND HEP 393
T-CELLS THERE IS NOT A LOT OF
SIGLEC BINDING. THIS IS 7
HOWEVER IF WE CO-EXPRESS ST85IA6
ALONG WITH CD4 4 YOU CAN SEE THE
SHIFT. SO IT INDICATES THAT CD4
4 BEING MODIFIED BY ST85IA6 IS
GENERATING A LIGAND FOR
RECOMBINANT SIGLEC 7. BECAUSE
ST85IA6 PUTS ON A SECOND SIALIC
ACID ON TO A PREEXISTING SIALIC
ACID WHAT COULD THAT FIRST
SIALIC ACID BE? HOW IS IT
ORIGINALLY PUT ON? SO WE DECIDED
TO LOOK AT ST 3 GAL 1 AND FT 6
GAL 1. SO HERE WE CO-TRANSFECT
CD4 4 WITH ST 3 GAL 1 WE DO SEE
THERE IS ENHANCEMENTS OF BINDING
OF RECOMBINANT SIGLEC 7. WHAT WE
SEE IS BOTH ARE TOGETHER. SO ST
3 GAL 1 ALLEN AN BOTH TOGETHER
YOU CAN SEE SYNERGY TO GENERATE
LIGANDS FOR SIGLEC 7. THIS IS
SPECIFIC FOR SIGLEC 7 IF WE USE
ST 6 GAL 1 WE DON'T SEE ANY
SYNERGY BETWEEN ST85IA6 ALONE
ABOUT ST85IA6 PLUS ST 6 GAL 1.
WHAT MY SLIDES WERE
AUTOMATICALLY FLIPPING THROUGH
IS THAT IT DID NOT GENERATE ANY
LIGANDS FOR RECOMBINANT SIGLEC
9. SO JUST TO SUMMARIZE IS THAT
WE IDENTIFIED THAT ST85IA6
GENERATES OR SYLATES CD4 4 OR
MURINE TUMOR CELLS, USING 293
CELLS WE CAN ST85IA6 CYLATION OF
CD4 4 LEADS TO GENERICS OF
LIGANDS FOR SIGLEC 7 BUT NOT 9
AND GENERATION OF LIGANDS FOR
SIGLEC 7 WAS ENHANCED WHEN ST 3
GAL 1 WAS CO-EXPRESSED WITH
ST85IA6 ALONG WITH CD # 4. SO
WITH THAT I WOULD LIKE TO THANK,
I DONE DO MASS SPECTROMETRY SO
WE REALLY DEPEND UPON
(INAUDIBLE) FOR DOING THE HARD
WORK TO IDENTIFY POTENTIAL
TARGETS AND THE WORKEN ST85IA6
IN THE LAB WAS STARTED BY DAVID
FRIEDMAN, RECENTLY DEFENDED AND
MOVED ON, HE WAS ONE LOOKING AT
ST85IA6 IN CANCER AND THE
IDENTIFICATION OF WHAT ARE THE
TARGETS THAT ARE BEING MODIFIED
BY ST85IA6 GENERATES LIGANDS FOR
SIGLECS HAVE BEEN TAKEN UP BY
ANOTHER GRADUATE STUDENT IN THE
LAB MEGHAN CHEN. SO WITH THAT
THANK YOU SO MUCH FOR YOUR
ATTENTION. I WOULD BE HAPPY TO
TALK TO ANYONE AFTERWARDS AT THE
BREAK ABOUT THE WORK THAT WE ARE
DOING. THANK YOU.
[APPLAUSE]
CYLATION
>> NEXT SPEAKER IS DR. DRAKE,
SIMPLIFIED GLYCAN WORK FLOWS.
>> THANKS TO THE COMMON FUND FOR
GIVING THIS TALK. ON BEHALF OF
MY COLLEAGUES, -- I SEE THE
SLIDE THAT -- THERE WE GO. MY
COLLEAGUE, IS MULTI-PI METHA AND
PEGGY ANGEL, WE ARE APPRECIATE
THE LAB ALLOWED US TO MAKE THESE
TOOLS. THE GRAND WAS SUCCESSFUL,
WE HAVE BEEN VERY HAPPY WITH IT.
BECAUSE WE STARTED WITH THE IDEA
WE WORK WITH CLINICAL SAMPLES,
COULD WE HAVE A METHOD, METHODS
WHERE WE CAN LOOK AT ANYTHING WE
CAN GET CLINICALLY WHETHER
TISSUE, BLOOD SERUM, URINE,
BIOFLUIDS YOU NAME IT. THINGS
READILY AVAILABLE BUT THEN
GLYCOTYPE ESSENTIALLY. SO THIS
WAS ADAPTED FROM OUR METHODS
DOING TISSUE IMAGING. AND THE
FACT THAT WE HAD TISSUE ON A
SLIDE. WE SPRAY THE TISSUE WITH
PENGASE TO RELEASE THE LIGANDS
SO WE HIT WITH MULTI-LASER AND
DETECT WHAT IS PRESENT IN EACH
SPACE. SO USUALLY WE WERE USING
THESE IMAGING WORK FLOWS, WE ARE
GOING TO SKIP THAT. TO DO THAT
AND THEN GENERATE THE SPECTRA
AND WE CAN GENERATE THE
ESSENTIALLY HEAT MAPS OF
INTENSITIES WHERE THE DIFFERENT
GLYCANS WORK. YOU CAN ALSO ADAPT
TO THINGS NOT TISSUE, OTHER
THINGS ON THE SLIDE. AND
ANTIBODY ARRAY, WHAT IF WE SPOT
SERUM, THINGS LIKE THAT. SO WE
HAVE PULLED OFF MOST OF THIS
NOW, STARTED WITH TISSUE, I WILL
SHOW EXAMPLES OF WHAT WE ARE
DOING WITH WORK FLOWS ON
CULTURED CELLS. THAT PEGGY ANGEL
DEVELOPED. AND MEHT SPEARHEADED
THE ANTIBODY ARRAY ACTIVITIES.
AND I HAVE SPENDING TIME ON
BIOFLUIDS JUST PROFILING THE
CLINICAL FLUIDS. THIS WAS NOT
ACTUALLY PART OF THE COMMON FUND
PROPOSAL AT THE TIME BUT
EVERYTHING WE DEVELOPED WITHIN
THE COMMON FUND SPUN NATURALLY
IN TO THIS SO IT WAS A NICE
BY-PRODUCT. WE WILL SKIP
THROUGH. SO WE ALWAYS HAD THE
PREMISE USING A MALDI INSTRUMENT
BASED ON HOW SIMPLE BENCH TOP
MALDI TRANSFORMED CLINICAL
MICROBIOLOGY. GLYCOTYPING OR
BIOTYPING OF BACTERIA IS DONE
NOW BY MALDI, VERY ROUTINE,
ESSENTIALLY A BAR CODE TYPE.
COULD WE DO THAT WITH GLYCANS
BECAUSE ESSENTIALLY THE MALDI IS
GIVING YOU THAT INFORMATION,
VERY CONSISTENT N GLYCAN PEAKS,
KNOWN MASSES, CAN WE DO THIS FOR
DIFFERENT CLINICAL CONDITIONS.
WE WON'T GO IN ORDER OF THE AIMS
BUT PEGGY PUBLISHED THE INITIAL
PAPER, THE SHORT VERSION YOU CAN
GROW ON THE SLIDE, ANY ATTACHED
CELL ON THE SLIDE WE CAN SHOOT
IT AND DO GLYCOPROFILING. IT'S A
METHOD PAPER AND DOESN'T TAKE
THAT MANY CELLS TO GET GOOD
SIGNAL, WE CAN GO LOW AS 10
DEPENDING ON CELL TYPE. WE ARE
GETTING TOWARD SING CELL, BASED
ON PEGGY'S CCRC ROOTS SHE ALSO
ADAPTED THE IDOL METHOD
INCORPORATING GLUTAMINE INTO THE
CELLS. I LOVE THIS BECAUSE YOU
CAN JUST SEE THE ONE IN YELLOW
REPRESENT THE HIGH MAN NOSE
INCORPORATED, WHILE BLUE CELLS
AREN'T INCORPORATING -- THAT
DOESN'T MEAN THEY ARE DEAD BUT
THEY ARE NOT CYCLING PROBABLY.
SO INTERESTING INFORMATION THERE
LOOKING AT GLYCANS. THIS ISN'T
A SINGLE CELL IT IS A SINGLE
ORGANISM BUT METHODS DEVELOPED
TO DO THE CELLS WE WORK WITH
HEATHER, STEAT AND RICH STEAT
ALSO IN SOUTH CAROLINA GREEN
WOOD KINETIC CENTER. THEY HAD
ZEBRAFISH MODEL OF A CDG FOR
PHOSPHOMANU MYTASE SO TOOK A RUN
AT IT, LOVE PICTURES BUT THERE
IS BIOLOGY HERE, IT IS WHAT YOU
EXPECT HIGH MANNOSE ARE
SUPPRESSED AND THE PHOSPHOMYTASE
MUTANT BUT FOR SOME OTHER REASON
IN THAT MUTANT THE LITTLE BY
SECT GLYCANS ARE VERY ELEVATED
SO SOME METABOLIC CONVERSATION
GOING ON THERE. BRIEFLY, I HAVE
A STUDENT WHO DECIDED TO LOOK AT
THREE PANCREATIC CANCER CELLS
LINES, ONE CONTROL, FOUR DRUG
TREATMENTS THREE TIME POINTS,
WITH HEAVY GLUTAMINE. ONCE HE
SORTS THIS OUT HE GRADUATES,
NICE PAPER TOO BUT AMAZING
THINGS, WE HAVE 80 GLYCANS TO
SORT THROUGH ACROSS CELL LINES
AND TIME POINTS. THERE WAS SOME
INTERESTING YOU LOOK
INTERESTINGLY THIS ONE IS
CONTROL. ONE EXAMPLE OF GLYCAN
ELEVATED IN RESPONSE TO DRUGS.
MOST PROFILE LIKE THIS THERE IS
A LAG BEFORE REINCORPORATING IT.
SO THAT IS ONGOING. THAT IS ONE
TOOL. IN THE CELLS. THE OTHER
ANTIBODY PANELS PUBLISHED AND
STANDARDIZE AND CURRENT
PROTOCOLS ALSO. AND THEN AS
PART OF THE GRANT WE ARE LOOKING
AT PRIMARILY THE IMMUNOGLOBULIN
AND THE SUBTYPES AND SAME
INFORMATION. THIS IS GOING TO BE
FOLLOWED UP BY THE NEXT SPEAKER,
BEFORE THE BREAK, THAT
COMMERCIALIZED THIS. SO THE IDEA
IS THAT AGAIN IF YOU PUT
ANTIBODY ARRAY ON A SLIDE, THEN
DO SAME WORK FLOW INCUBATE
CAPTURE THE TARGET. WE KNOW WHAT
IS IN EACH SPOT. IF ANTIBODY IS
GOOD WE KNOW WHAT TARGET IS.
THESE TARGETS BEING
GLYCOPROTEINS IN THIS CASE
IMMUNOGLOBULINS, IMMUNOGLOBULIN
Gs, AND WORK FLOW YOU SPRAY
THE PENGASE YOU CAN DETECT
GLYCANS NICE PAPER PUBLISHED,
LAST THREE OR FOUR MONTHS, WE
TURN GLYCOFIBER TYPIER. THIS IS
BLURRY BETWEEN ACADEMIC HATS AND
COMPANY HAT. THIS WE WILL
COMMERCIALLYZE FOR EARLY LIVER
DISEASE. IT IS LOOKING AT EARLY
STAGE FIBROSIS, EARLY STAGE
DISEASE FROM LATER STAGE
FIBROSIS. THIS IS THE THING WE
DO WITH EVERY ANTIBODY, THE
BEAUTY OF GOING WITH THE
ABUNDANT SERUM GLYCOPROTEIN,
STANDARDS ARE THERE. SO YOU CAN
LOOK AT STANDARD, THEN YOU CAN
USE NORMAL SERUM TO CHARACTERIZE
WHAT PROFILE ARE YOU GETTING. SO
STANDARD IS NOT FROM NORMAL
SERUM BUT IT IS CONSISTENT THIS
WAY. WHEN YOU LOOK AT CLINICAL
SCENARIO, THIS IS YOU SEE
SUBTYPE DIFFERENCES. GLYCANS
SUBTYPE YOU CAN LINK BACK TO
CLINICAL CONDITION. YOU CAN
START TO GET RAW CURVES THAT
HAVE VERY SIGNIFICANT CLINICAL
MEANING. UNFORTUNATELY THIS WAS
ONLY DONE ON 80 SAMPLES SO THIS
IS SET UP FOR MUCH LARGER
PROCESS. WE'LL COME BACK IN A
BIT. NEXT STEPS ARE TWO
DIRECTIONS, ONE I WILL COVER
SHORTLY. ABOUT HAVING SPUN THIS
OUT COMMERCIALLYLY. ALSO WE
ENVISION ON SIMPLE ANGLE TAKING
THE NEXT TOP TENSER RUM
GLYCOPROTEINS INCORPORATED INTO
THE WORK FLOWS. SO THAT WAS --
THE LAST THING THIS ONE TOOK A
WHILE AND WE WENT ABOUT IT IN
DIFFERENT WAYS. THE IDEA IS DO
IMMUNE CAPTURE IN IMMUNE CELLS
AND GLYCOTYPE IT THE SAME WORK
FLOW ON THE BACK END. IT TOOK
SOME TIME AND DEDICATED GRADUATE
STUDENT JAKE DRESSMAN TO PULL IT
OFF BUT WHAT YOU ARE SEEING IS
DOES LOOK LIKE LYMPHOCYTES
HALFWAY TO YOU AND THEY ARE
ATTACHED, ON A SLIDE BECAUSE
THEY ARE ANTIBODY CAPTURED.
TURNED OUT THE KEY WAS THE
FIXATION DELIPID DAVIS STEP WE
DO A DELIPIDATION TO GET RID OF
THE SMALL MOLECULES AND SOME OF
THE SALTS THAT WASH WITH IT.
OPENS UP GLYCANS FOR ACCESS. IN
THIS CASE THE KEY WAS AFTER YOU
INCUBATE CELLS CAPTURED THEN DID
FORMALIN FIXATION TO LOCK IN.
ONCE WE DID THAT, IT'S GOLDEN,
WE HAVE DONE THINGS WITH FACT
SORTED CELLS YOU CAN APPLY THAT
AND GIVE SAME WORK FLOW SO
APPEARING QUITE ROBUST. WE ARE
STILL FRONT END BUT I WILL SHOW
YOU QUICK EXAMPLES. WE CAN ALSO
DO -- WE DID NON-LYMPHOCYTES,
HEPG 2 LIVER CELLS TO SHARE YOU
CAN DO THIS CAPTURE METHOD AND
TREATED WITH 2 FOLLOW -- DEOXI
DEOXIFOLLOW FUCOSE. AND YOU CAN
SEE INVERSE OF THE TREATED ONES,
LOSE THAT EXTRA FUCOSE AND ONES
THAT DON'T YOU CAN SEE BUILDING
UP IN THE ONE THAT WASN'T -- THE
INVERSE. YOU TELL I DIDN'T DO
THIS ONE. WE HAVE DONE GREAT
PROOF OF CONCEPT BEFORE GETTING
TO PBMCs, TAKING SPLEENS OR
T-CELLS LYMPHOCYTES OUT OF MOUSE
SPLEENS AND SORTING THEM, GOING
AFTER THE CD4 AND CD8 IN THAT
POPULATION. AGAIN YOU CAN SEE
WHAT I LIKE ABOUT THIS ASSAY,
MAKES ME SMILE IS YOU CAN SEE
THIS CAPTURED PUT IT UNDER SCOPE
AND SEE IF YOU GOT A CELL THERE.
SO HE CAN AGAIN LOOKING AT THE
CD4 AND CD8 PROOF OF CONCEPT IS
CLEAN. IT IS THE TYPE OF GLYCANS
WE SEE ACTIVELY GROWING
LYMPHOCYTES, THE MICE SUCKING
ONE Z AS WELL AS HIGH MANNOSE.
THE CBs ARE IN PLAY BUT THEY
ARE GETTING GOOD. WE ARE
GETTING INTO THE LESS THAN 10
FOR THE MOST PART SO WE LIKE
WHERE THIS ASSAY IS GOING. FOR
THE MOST PART. THEN GIVING
CREDIT HE ADAPTED A CELL
COUNTING PROGRAM ADAPTED TO
THIS. SO AGAIN THE ASSAYS WE ARE
GOING TO HAVE INTENSITY DATA
MORALIZE CELL COUNT SO EXTRA
LAYER OF QUANTITATION, IS GOING
B TO INTERESTING DOWN THE LINE.
SO THERE IS A LOT YOU CAN DO
WITH THIS THINGS, LOT OF THE
TARGETS SO WE WILL GO TO BLOOD
AND PBMCs FROM CLINICAL
SAMPLES, THEY ARE WORKING ON
ORGAN ASSOCIATIONS WITH THE
MOUSE, REALITY IS, PRETTY MUCH
3-D ASSAYS WE DISCUSSED FOR THE
MOST PART, IT IS ALL GOING TO GO
TO IMMUNOTHERAPY. DOWN THE
LINE, THAT IS WHERE WE APPLY
THESE. I WANTED TO THANK COMMON
FUND, WE HAVE ALL THESE WORK
FLOWS, WE HAVE TURNED INTO A
PLATFORM THIS IS ESSENTIALLY A
PLATFORM TECHNOLOGY NOW WHATEVER
WE CAN STICK ON WHATEVER WE CAN
STICK ON GLASS THIS BACK END IS
A PLATFORM WE KNOW WE CAN
GENERATE THE GLYCANS OFF OF IT.
THE GOAL BEING WE WANT TO DO
PATIENT SPECIFIC GLYCOTYPE OR
TEST FOR STRESSING CANCER WORLD
YOU CAN SEE WE GET ALL THESE
TYPES OF SAMPLES. AND GLYCOTYPE
IT. BRIEF OUTREACH COMPONENTS,
DURING THE PANDEMIC WE STARTED
COLLABORATION THAT FORM
GLYCOGLYCOMICS CENTER OF
EXCELLENCE WITH THE
MANUFACTURERS OF THE MASS SPEC
WE USE. WE ARE DEDICATING THIS
FOR TRAINING, TO TRY THE GET
ASSAYS OUT INTO OTHER PEOPLE'S
HANDS. AND IN THAT REGARD, THE
NUMBER AFTER HURRICANE SEASON IS
OVER ANYBODY WANTS SOME HANDS ON
RAINING WITH ANY ONE OF THESE
TYPES OF APPROACHES, WE ARE
GOING TO DO A SMALL GROUP
IMAGING WORKSHOP HANDS ON
METHODOLOGY THAT CHARLESTON
CHARLES DID IN OUR PLACE. WHERE
WE ARE GOING CONTINUED OUTREACH,
AGAIN, THE IDEA OF GETTING TOOLS
OUT THERE, THAT COLLABORATIVE
CENTER HELPS ON THE TRAINING
PART, WE HAVE BEEN WORKING WITH
GLYGAN ON PROJECTS, KIOKO ON
DIFFERENT GLYCO 2 CAN, WORK FLOW
PUSH BUTTON FOR ACCESS TO
GLYCANS AN COMPANY IS ALSO
WORKING ON OUTREACH PART. A LOT
OF TOOLS WE ARE GOING TOWARD NIH
TISSUE MAPPING INITIATIVES,
TRYING TO INTEGRATE ALL THIS
GLYCOMIC IN SINGLE CELL OMICS IN
THESE CLINICAL SAMPLE TYPE
PROJECTS. AND REALLY EXPAND ON
THE CLINICAL BIOFLUID ANGLE. WE
ARE WORKING WITH PARTNERS AT FDA
ON COVID SAMPLES, AND THEN GOT
THIS PLATFORM CHANGE ENZYME,
CHANGE SIGNAL, IF WE DON'T USE
PENGASE WE HAVE THIS PLATFORM IN
PLACE TO LOOK FOR OTHER TARGETS.
WE HAVE FASCINATING DATA THAT
REMOAN SIMON DEVELOPED USING
AMYLASES AND GLYCOGEN,
FASCINATING STUFF AND TISSUES.
STACY MALAKE MENTIONED THE
GLYCOPROTEASES MUCINASES ARE
STABLE AND JUST PERFORM
EXCEPTIONALLY WELL ON TISSUE.
GOT REALLY NICE DATA MANY THAT
REGARD.REGARD. , THEN TALKING WITH
CHRIS NEW ENGLAND BIOLABS, HE'S
GOT A LOT OF BELIE COST DAYSES,
SOME COMMERCIALLY AVAILABLE AND
SOME AREN'T WHEN STRIKING
PARTNERSHIPS TO START
INTEGRATING THIS IN TO THESE
DIFFERENT WORK FLOWS. AND SEE
WHERE IT GOES. A LOT OF
ACKNOWLEDGMENTS. WE HAVE
PRIMARILY STUDENTS WHO HAD DONE
A LOT OF GREAT WORK. AND MY
COLLEAGUES PEGGY AND THE COMMON
FUND AND ANAND, AND COMMON FUND
FUNDING. NOW, FOR THE NEXT
SPEAKER -- BEFORE THE NEXT
SPEAKER. I'M HAPPY TO INTRODUCE
THE NEXT SPEAKER, SEEMS LIKE I
HAVE KNOWN HIM A LONG TIME. YEAH
OKAY IT IS ME. SEE, I CHANGED.
YOU WON'T GET THE PITCH, MAYBE A
LITTLE BUT THIS IS HOW WE ARE
TRYING TO COMMERCIALIZE AIM ONE,
THE GLYCAN ANTIBODY ARRAYS.
ANAND ALWAYS TO HIS CREDIT HAS
BEEN A LIVER DISEASE PERSON. I
DON'T HAVE TO FOCUS, I CAN DO A
LOT OF DIFFERENT ONES. I ALWAYS
LOOK FOR THE NEXT TISSUE. THAT
IS HOW WE ARE GOING TO TARGET
THAT AGAIN WITH SERUM IS IDEAL
FOR LOOKING AT LIVER DISEASE, IT
IS ALWAYS THERE. AND IRONICALLY
WE ARE LOOKING AT
IMMUNOGLOBULINS BUT THE
HEPATOCELLULAR CARCINOMA IN
LIVER DISEASE IN GENERAL IS
BUCKLING ALL THE TRENDS AS FAR
AS DECREASING INCIDENCE. IT IS
GETTING WORSE AND ANAND WAS AT A
MEETING THIS WEEK AND IT IS
ACCELERATING SO THERE'S ALL
RIGHT REJECTIONS ABOUT LIVER
CANCER INCIDENCE BUT IT IS
GETTING WORSE QUICKER. AND WE
ARE ALL GLYCOBIOOLOGISTS HERE,
USUALLY WE THINK LIVER CANCER
AND HEPATITIS B OR C IS ALWAYS
PART OF THE GLYCO COMMUNITY, IT
IS FATTY LIVER NOW. IT IS STILL
ENDS UP GETTING TO THE SAME
PATHWAY AS FIBROSIS CIRRHOSIS
LIVER CANCER BUT MOST OF THE
INCIDENCE ESPECIALLY IN U.S. AND
LOT OF EUROPE IS NOW FATTY LIVER
AND THAT IS WHERE WE ARE
TARGETING ASSAYS. THE TEAM IS
MYSELF ANAND AND PEGGY, FORMER
GRADUATE STUDENT DANIELLE WE
WERE FOUNDERS. ANYONE WE KNEW A
COLLEAGUE FROM THE IMAGING
COMMUNITY STEVEN WHO IS 25 YEARS
AT GLAXOSMITHKLINE IN PKPD SO HE
IS A GOOD ASSAY DEVELOPER THAT
WE HAVE GONE ON FOR -- HE
RETIRED THEN THE NEXT DAY WE GOT
INVOLVED. IS HOW THAT WORKED OUT
SO WE ARE USING ANN BODY ARRAY,
THERE IS A LOT OF GOOD ASSAY,
BUILDING ON THE LONG HISTORY O
OF LOOKING AT IMMUNOGLOBULIN G
GLYCOSYLATION, WE DIDN'T HAVE TO
INVENT THAT PART THERE'S A LOT
OF STUDIES OUTS THERE AND
SAMPLES THAT HAVE GONE THROUGH,
MAINLY IN EUROPE BUT ANAND WAS
PART OF EARLY STUDIES IN THAT
REGARD TOO. SO REALLY WHAT WE
HAVE SEEN IS WE WANT TO MAKE IT
-- THERE IS THE PITCH PART, EASY
FASTER COST EFFECTIVE AND
BETTER, THAT'S CURRENT COST
BETTER THAN CURRENT ONES BUT IT
COMES DOWN THE BAR CODE CONCEPT,
THE -- I LIKE TO THINK OF IT THE
IMMUNOGLOBULIN G GLYCANS AS
GOLDILOX TYPE ONES. THESE ARE
JUST RIGHT ONES, RIGHT? THE ONES
THAT THERAPEUTIC COMPANIES HAVE
TO HAVE ON THEIR ANTIBODY SO
THEY DON'T WORRY ABOUT IT AND
THEN THERE IS THE OTHER ONES.
THAT CAUSE PROBLEMS. THAT IS
REALLY MEASURING, WE DID HAVE
THIS PUBLISHED THIS IS LEADING
TO FUNDING WE HAVE GOT ANOTHER
PHASE 1 THAT STARTED MONTH AGO.
IF YOU CAN DO EARLY TO EARLY
LIVER DISEASE LATE STAGE
FIBROSIS, YOU CAN ALSO DO
CONTINUED ACCESS TO LIVER CANCER
HCC. SO WORKING ACROSS THAT
WHOLE SPECTRUM. AND WE HAVE
REALLY GOOD SEED FUNDING FROM
BRUKER, OWNERSHIP IN THE
COMPANY, SO WE WENT FROM LLC
WHICH LAST TIME WE HAD A MEETING
WE WERE LLC TO INC BECAUSE WE
HAD TO. SO INTERESTING PROCESS
ON ITSELF. GETTING BACK TO THE
TOOL PART. PART. WHAT WE ARE DOING
WITH SBIR IS REALLY OPTIMIZING
THE SLIDE CHEMISTRY SO THE SLIDE
CHEMISTRY P CAPTURE ANTIBODY,
AMOUNT OF ANTIBODY, QCING IT TO
THE END, APPARENTLY YOU DO
CLINICAL ASSAY SUPPOSED TO LOOK
THE SAME EVERY TIME YOU USE IT.
WHO KNEW. KIDDING.KIDDING. KIDDING. KIDDIN G. ANYWAY WE
ARE WORKING ON THE QC PART BUT
THAT SPINS OFF TO OTHER
TECHNOLOGY BECAUSE WE GO WITH, G
PANELS BUT CAN INCORPORATE OTHER
ANTIBODY TARGETS. THIS
POTENTIALLY COULD BE SOMETHING
WE SELL FRANKLY AS FAR AS ARRAY
FOR THIS. AND WE ARE WORKING ON
DATA ANALYSIS AND ALGORITHM SIDE
AND OUTSOURCING THESE WITH
DIFFERENT COMPANIES TO TO GET
THAT SO CLINICAL READ OUT PART.
BUSINESS MODEL WISE IS LINKED TO
THE PROGRAM. WE ARE NOT
TARGETING GLYCOBIOLOGIST FOR THE
ASSAY. THESE ARE CLINICIANS AND
CLIA LABS. FARTHEST AWAY YOU
CAN GET FROM GLYCOBIOLOGISTS, SO
WE WILL SEE HOW THAT GOES. THIS
IS THE LITTLE BENCH TOP THAT IS
SMALL, MUCH SMALLER THAN THIS
PROTEOME. WE CAN DO SERUM
PROFILING. JUST TOTAL GLYCAN
PROFILE SO THE LITTLE BENCH TOP
ONE COMPARED TO THOUSAND TIMES
BETTERS ARE SOLUTION SMART FLEX.
IT IS MORE SATISFYING, DOESN'T
MATTER WHICH IS IT, WE ARE HAPPY
WITH THIS. IN PRINCIPLE THIS
BENCH TOP CAN BE USED FOR BAR
CARD PROFILING. WE DO MARK THE
KIT TO DO IMAGING BUT THAT WAS
MAINLY BECAUSE WE ARE PART OF
THE KIT ANYWAY WHEN WE DID THE
CLINICAL ASSAY. SO WE HAVE SOLD
A FEW OF THOSE. IT IS NOT THE
BUSINESS MODEL. AND WE DO HAVE
CUSTOMIZE ARRAYS COMING DOWN THE
PIKE WHICH BOTH OF THESE MAY HIT
THE MORE COMMERCIAL SPACE AND
THAT IS CLINICAL ALSO IN SPIRIT
OF COMMON FUND. I WILL STOP
WILL.
[APPLAUSE]
I WILL STOP THERE.
>> GREAT.
[APPLAUSE]
>> SO WE HAVE A SHORT BREAK. WE
COME BACK HERE AT 3:50. AT 3:50
DR. EDWARDS IS HERE. HE WILL
GIVE HIS TALK.
>> OUR NEXT SPEAKER IS DR.
NATHAN EDWARDS AND HE'S GOING TO
TALK ABOUT THE WORK THAT HE AND
DR. GOLDMAN HAVE BEEN WORKING
ON.
>> I WANT TO TALK TO YOU ABOUT
THE WORK THAT ROD AND I WORKED
ON TO TRY TO PROVE OUT LCMS
ANALYSIS STRATEGY FOR SITE
SPECIFIC PROTEIN GLYCOFORMS. THE
PROMISE HERE THAT WE SAW TO
REALIZE WAS THIS IDEA THAT WE
COULD RUN A DIA STAR WORK FLOW
FOR QUANTIFICATION OF
GLYCOPEPTIDES USING THE SAME
ANALYSIS STRATEGY BEING
SUCCESSFUL FOR PROTEOMICS.
ENABLING US TO GET SENSITIVE
COMPREHENSIVE QUANTITATION OF
GLYCOPEPTIDES AND DR. BY
UNDERSTANDING NOT ONLY WHAT
GLYCANS ARE OBSERVED BUT WHERE
ON PARTICULAR PROTEIN. RON'S
LAB IS RESPONSIBLE FOR PROTOCOLS
AND ANALYTICAL WORK FLOWS OF THE
PART OF THIS ANALYSIS. MY LAB
IS RESPONSIBLE FOR INFORMATICS
SIDE OF THINGS. TRYING TO
CONSTRUCT GLYCOPEPTIDE SPECTRA
AND TRANSITION LIBRARIES WHICH
ARE NECESSARY FOR THE DIA
ANALYSIS. AND AS A SIDE BENEFIT
OF THAT, WHAT WE HAVE IS QUITE A
SIGNIFICANT LIBRARY OF
GLYCOPEPTIDES WITH THIS SPECTRAL
EVIDENCE. THAT PROVIDE SPECIFIC
DIRECT EVIDENCE OF EXISTENCE OF
THESE GLYCANS AN SPECIFIC SPOT
ON A PROTEIN, WITH ENOUGH
EVIDENCE YOU CAN DECIDE WHETHER
YOU BELIEVE THEM YOURSELF. THIS
IS WEBSITE THAT IS CONSTRUCTED
TO DAYS PLAY THIS
INFORMATIONMENT -- DISPLAY IN
INFORMATION. GPT WIKI IS THE
THING TO GOOGLE THAT COMES UP
NUMBER ONE IN SEARCH RESULTS. RESULTS.
WE HAVE TWO PRIMARY
COMPREHENSIVE ANALYSIS THAT WE
INTENT TIME ON, SERUM IS THE
FIRST FIRST, A GOOD SUBSTRATE AT
GLYCOPEPTIDE ABUNDANCE AND HEP
293 CELLS. WE HAVE TARGETED
ANALYSES WHERE SPIKE PROTEIN
FROM COVID AND HUMAN ACE TWO
PROTEIN ANALYZE DIRECTLY IN
ORDER TO LOOK AT THEIR
GLYCOSYLATION. NOT SO MUCH A
COMPREHENSIVE NUMBER OF PROTEINS
KIND OF ASSAY BUT AS YOU CAN SEE
NUMBER OF WHITE LANDS SERVE TWO
SPECIFIC PROTEINS WAS QUITE
HIGH. LASTLY, WE HAVE THE NIST
LIBRARY AND WITH THEIR
PERMISSION ADDED EACH INTO THE
DATABASE FOR OUR REFERENCE.
THESE GLYCOPEPTIDES ARE STUDIED
FIRST USING TYPICAL
FRAGMENTATION ANALYSIS WHERE WE
LOOK AT EDD HCC SPECTRA AND
TRIGGER HCC SPECTRA AFTERWARDS.
THE PAIR OF SPECTRA REPRESENT
COMPLIMENTARY INFORMATION WHICH
TOGETHER GIVE MORE CONVERSATION
IN THE CORRECTNESS OF
IDENTIFICATION. THIS IS
EXTENSIVELY ORGANIZED SET OF
INFORMATION, HERE IS SAME
INFORMATION I TOLD YOU ABOUT.
LESS REGULATION THAT OF SAMPLES
WITH DIAND AND GLYCOPEPTIDES ARE
OBSERVED USING DIA ANALYSIS AN
TRANSITIONS AND THEIR EXTRACTED
CHROMATOGRAMS ARE AVAILABLE IN
THIS DATABASE FOR VIEWING. I
CAN'T CLAIM GPT WIKI SET THE
WORLD ON FIRE, WE ARE NOT
PULLING IN THOUSANDS OF VISITORS
A DAY BUT STEADY USE OF TWO
YEARS, WE HAVE THE BASELINE
LEVEL OF USE, WHERE PEOPLE ARE
VISITING THE SITE AND USING IT
TO UNDERSTAND WHAT IS GOING ON.
EXTENSIVE USE AROUND THE WORLD
THOUGH U.S. IS DOMINANT SITE OF
USE. THE GLYCOPEPTIDES AND
GLYCANS AS A SPECIFIC SITE ON
PROTEIN HAVE BEEN INTEGRATE WITH
THE GLYCAN INFRASTRUCTURE. YOU
CAN SEE ANY OF GPT WIKI EVIDENCE
WE HAVE, DISPLAYED IN GLYGEN AND
OTHER SOURCES OF EVIDENCE
PROVIDED FOR GLYCANS ON SITES IN
GLYCOGEN THESE LINKS PROVIDE
ACCESS TO THE SPECTRA WHICH
LABELED SPECTRA AT THAT, WHICH
GIVE YOU THE EVIDENCE FOR WHY WE
BELIEVE THAT GLYCAN IS THERE ON
THAT PROTEIN. I WANT TO GIVE
SOME IDEA HOW THE DATA ORGANIZED
AFT HOW YOU CAN FIND WHAT IS
THERE. WE HAVE A PAGE FOR EACH
PROTEIN WHICH INDICATES SAMPLES
THAT IS OBSERVED ON AND SITES
THAT HAVE GLYCOPEPTIDES ATTACHED
AS WE CAN SEE THE ASPARAGINE 131
IS BY FAR MOST NUMBER OF
GLYCOPEPTIDES OBSERVED. AND
WHEREAS THE OTHER TWO SITES ARE
LESS ABUNDANT OR LESS VARIETY OF
GLYCOPEPTIDES OBSERVED.
INDIVIDUAL TRANSITION GROUP
PAGESES INDICATE EVIDENCE FOR A
SPECIFIC GLYCOPEPTIDE AT SPOT ON
PROTEIN. WE ARE IDENTIFYING
THINGS AT LEVEL OF COMPOSITION
BUT IN ANALYSIS PIPELINES TO
PROVE OUT AND COLLECT EVIDENCE
FOR GLYCANS WE ARE ANALYZING
MULTIPLE STRUCTURE EACH WHICH
HAVE COMPOSITION SO WE CAN
ACCOUNT FRAGMENTS SPECIFIC TO
PARTICULAR TOPOLOGY VERSUS THOSE
THAT ARE COMMON TO ALL STRUCTURE
THAT HAVE THAT COMPOSITION.
CHROMATOGRAM FOR THIS
GLYCOPEPTIDE, WHICH AGAIN IS
HELPFUL IN UNDERSTANDING THE
STRENGTH OF EVIDENCE AND WE
PRESENT THE PAIR OF SPECTRA HCD
AT THE TOP AND HCD WHICH
TOGETHER PROVIDE COMPLIMENTARY
INFORMATION FOR THE IDENTITY OF
THIS GLYCOPEPTIDE. IN
PARTICULAR THIS VIEWER IS
ZOOMABLE, YOU CAN ZOOM IN ON ANY
ASPECT OF THE SPECTRA,
PARTICULARLY USEFUL FOR STUDYING
THIS FRAGMENT HERE WHICH
PROVIDES EVIDENCE OF THE CORE
FUCOSYLATION ON THIS
GLYCOPEPTIDE. IMPORTANTLY I
WILL TALK ABOUT THIS IN A
MINUTE. THIS CHARGE REDUCED
PREPORKER IN ET ACD SPECTRUM IS
IMPORTANT FOR US UNDERSTANDING
WHETHER OR NOT WE HAVE CORRECT
ASSIGNMENT OF THE PRE-CURSOR FOR
THESE SPECTRA. HERE IS AN
EXAMPLE OF THE DIA ANALYSIS OF
THIS GLYCOPEPTIDE AS PART OF A
COMPREHENSIVE ANALYSIS FROM
SERUM. THIS IS DI ANALYSIS
CONDUCTED USING THE TRANSITION
LIBRARY FORMED FROM THE PEPTIDE
FRAGMENTATION SPECTRA. SOMETHING
I MEANT TO SAY BACK HERE IS PART
OF THE REASON WE COLLECT TWO
TYPES OF SPECTRA, THE FIRST
SPECTRUM IS IMPORTANT BECAUSE IT
HELPS US IDENTIFY THE
GLYCOPEPTIDE SUBSTRATE. AND THE
GLYCAN ITSELF, SECOND SPECTRUM
IS IMPORTANT BECAUSE HCD
SPECTRUM IS ONE WE WILL THEN USE
TO LOOK AT INTENSITY OF
FRAGMENTS IN THE DIA ANALYSIS.
WE OBSERVE IN PARTICULAR IN THIS
SO CALLED SOFT FRAGMENTATION
MODE, WE LOSE THE DOMINANT
FRAGMENT HERE THIS BLUE PEAK
CORRESPONDS TO WHAT WE CALL THE
YM FROM THE GLYCAN STRUCTURES.
WE EXPECT THIS FRAGMENT TO
DOMINATE AND IN DOING SO GIVES
PARTIAL INFORMATION ABOUT
STRUCTURE. WE NOTICE THERE'S
THIS MIDDLE STRUCTURE HERE THAT
CANNOT FORM CORRECT MASS AS
TRANSITION, BY OBSERVING THE
BLUE TRANSITION HERE, SO
ABUNDANTLY SOME INFORMATION WE
DON'T PROVE THIS IS THE THE CORE
FUCOSYLATION FORM BUT THESE
FRAGMENTS HERE WITH THE ASTERISK
INDICATE TRANSITIONS SPECIFIC TO
THE CORE FUCOSYLATED FORM OF THE
GLYCAN. I WOULD LIKE TO GIVE A
SENSE OF THE DIFFICULTIES
INVOLVED IN DOING THIS TYPE OF
WORK SUCCESSFULLY. THERE WAS
AWFUL LOT OF WORK THAT WENT INTO
THIS TO TRY TO COME UP WITH A
CONCEPT OF NORMALIZED RETENTION
TIME. AS RELATE TO THESE DIA
STRATEGY AND GLYCOPEPTIDE
ANALYSIS. WE TRIED USING THE
TRADITIONAL IRT PEPTIDES THAT
ARE COMMERCIALLY AVAILABLE FOR
GLYCOPROTEOMICS AND WE FOUND
THEY DON'T DISTRIBUTE WELL OVER
REGION GLYCOPEPTIDES DISTRIBUTED
OVER. BUT THEY FORM THEIR
INITIAL STRATEGY. OUR SECOND
WAS TO LOOK FOR GLYCOPEPTIDES
ABUNDANTLY OBSERVED AND WHERE
SPACE REASONED REASONABLY ACROSS
THE RETENTION TIME FROM FILE.
WE USE SELECTED COMMON ABUNDANT
GLYCOPEPTIDES AS NEXT ATTEMPT
ONE WE HAVE THEM TO NORMALIZE
DEAFENINGS TIME. TROUBLE IS
THOUGH IF YOU PROCEED WITH
FRACTIONATION APPROACH OR ANY
OTHER APPROACH TO SELECTIVELY
TARGET GLYCOPROTEINS. OR SPREAD
OUT OR MAKE THEM SUCH YOU CAN
ANALYZE MORE DEEPLY, YOU NO
LONGER HAVE ACCESS TO COMMON
ABUNDANT GLYCOPEPTIDES. SO AS
WE EXPLORED WAYS TO LOOK DEEPER
INTO THE GLYCOPEPTIDE PROFILE WE
DISCOVERED GLYCOPEPTIDES NO
LONGER AVAILABLE AND DIDN'T
ENCOMPASS THE ENTIRE RETENTION
TIME RANGE. WE HAD ENOUGH
GLYCOPEPTIDES WITH NORMALIZED
PRETENSION TIMES THAT WE CAN USE
WHATEVER WE CAN FIND. THIS
WORKS WELL, WE DO INITIAL DIA
ANALYSIS, WE DABBLE RETENTION
TIME CONSTRAINT WE TAKE
CONFIDENTLY IDENTIFIED
TRANSITIONS AND USE THEM TO
CREATE CORRECTION FACTOR FOR THE
NORMALIZE RETENTION TIME.
NORMALIZE RETENTION TIME MODEL
IS BUILT FROM THE DDA ANALYSIS
USING THIS REGRESSION MODEL,
THIS WORKED QUITE WELL, SIALIC
ACID IS IMPORTANT WE DISCOVERED
ME THIONEINE OXIDATION MAKES A
DIFFERENCE AND THE
MONOSACCHARIDES IN STRUCTURE. WE
BUILT A SECOND REGRESSION MODEL
FOR EACH PEPTIDE. FOR EACH
UNDERLYING PEPTIDE SU SUBSTRATE.
THIS WAS GOOD AND BAD. THE
PEPTIDE ITSELF IS THE PRIMARY
DETERMINANT OF THE RETENTION
TIME FOR CAT. WE NEED THESE
OTHER FACTORS IN THERE THERE.
UNFORTUNATELY IF WE DON'T
OBSERVE DIFFERENT FORMS OF
GLYCOPEPTIDES FOR UNDERLYING
PEPTIDE SUBSTRATE, WHICH DON'T
HAVE ENOUGH INFORMATION TO FIT
THIS MODEL PARTICULARLY WELL.
WE WORKED HARD TO USE EVERY TOOL
AT OUR DISPOSAL TO MAKE THIS
GLYCOPEPTIDE ID SUBSTRATE HIGH
QUALITY. WE CURATED A LIMITED
SET OF PROTEINS AND GLYCANS, WE
USED FDR FILTERING AT EVERY
PLACE IN ORDER TO TRY AND ENSURE
WE DIDN'T HAVE FALSE POSITIVES.
WE USE NORMALIZE RETENTION TIME
FIT TO MAKE SURE THAT THE IDs
WHERE WE EXPECTED THEM TO BE IN
TERMS OF THE PREVIOUS
IDENTIFICATION WE SAW. AND WE
CHECKED THINGS LIKE THE
RETENTION TIME TO MAKE SURE IT
HAD REASONABLE SHAPE. ALL THESE
THINGS WERE GOOD ENSURING WE HAD
HIGH QUALITY GLYCOPEPTIDE IDs
BUT ULTIMATELY DISCOVERED THAT
WAS NOT ENOUGH. BECAUSE THERE
ARE PERFECTLY MANY THINGS, MANY
GLYCOPEPTIDES WHICH
SIGNIFICANTLY AND IMPORTANTLY
NOT RANDOM BUT NEVERTHELESS
WRONG. WE FOUND THIS BECAUSE WE
STARTED USING TWO DIFFERENT
SEARCH ENGINES AND THEY GAVE
INCONSISTENT RESULTS FOR THE
SAME SPECTRUM. AND IT WAS REALLY
THIS PROBLEM, THE TWO
PRE-CURSORS BEING ONE SIALIC
ACID THAT KILLS YOU. BECAUSE YOU
CAN'T -- THERE IS NOTHING IN
THERE TO FIND A WAY TO RESOLVE
THIS IN MOST SEARCH ENGINES WE
TRIED. SO WHAT WE DISCOVERED IS
HELPFUL TO TAKE CARE OF THIS
PROBLEM. IT TYPICALLY HAS A
CHARGE REDUCED PRE-CURSOR
ISOTOPE CLUSTER. YOU CAN USE
THIS TO CHECK PRE-CURSOR FOR
IDENTIFICATION YOU HAVE, FITS ON
THAT MONOISOTOPPIC PEAK OF THAT
PRECURSOR ISOTOPE CLUSTER. IF
YOU HAVE THE INSTITUTION OF TWO
FUCOSE INSTEAD, IT SHIPS OVER
ONE AND NOT IN THE RIGHT SPOT.
IN OUR ANALYSIS, WHERE WE WERE
LOOKING HARD FOR GLYCOPEPTIDES,
WE FOUND N OF ONE SPECTRUM
DIDN'T HAVE PRECURSOR ISOTOPE
CLUSTER TO RECOVER, ONLY THE
FRAGMENTATION SPECTRA IN ETD
DID. WE ALSO REQUIRE IF WE GOT A
CLAIM OF FUCOSE PEPTIDE AN
GLYCOPEPTIDE IT DOESN'T HAVE YES
ION WHICH SUPPORTS THAT BLAME.
AFTER DOING THESE STEPS, ALMOST
ALL OF THE 2, 3, 4 FUCOSE FORMS
IN OUR DATABASE WERE ELIMINATED.
WE HAVE SIGNIFICANT DISJUST OF
ALL MULTIPLY FUCOSE INFORMED --
FUCOSYLATED FORMS THAT I OBSERVE
IN VARIOUS ANNOTATION
SUBSTRATES. HAPPY TO TALK MORE
IF YOU HAVE QUESTIONS. THIS IS
UP AND WE ARE HOPING THAT IT IS
USEFUL TO THE COMMUNITY. IF YOU
HAVE A DATA SET THAT YOU THINK
MIGHT BE WELL DISPLAYED IN THIS
SETTING, IT HAS A CLEAR PATH TO
GET INTO GLYGEN AS ANNATIONS
WITH SUPPORTING SECOND --
ANNOTATIONS WITH SUPPORTING
SPECTRASPECTRA. HAPPY TO TALK TO YOU TO
FIGURE HOW TO GET INTO THAT TA
INFRASTRUCTURE. THANK YOU.
(OFF MIC)
(NO AUDIO) UO 1 GRANT FOR ALL OF
YOU IN PERSON, I FIRST WANT TO
THANK CARL PAM AND AMANDA AND
DOUG FOR THE LEADERSHIP AND
SUPPORT FOR THIS PARTICULAR
PROGRAM. SO AS MANY OF YOU KNOW,
WE HAVE BEEN DOING AND SPENDING
QUITE A BIT OF EFFORT TO DEVELOP
NEW TOOLS FOR FACILITATING HIGH
THROUGH PUT GLYCAN AND
GLYCOPEPTIDE QUANTITATION. FOR
THIS AUDIENCE PERHAPS NOT SO
NECESSARY TO REALLY INTRODUCE
THE IMPORTANCE OF GLYCOSYLATION
WHICH IS AMONG MOST IMPORTANT
COMPACT CHEMICAL MODIFICATION
FOR PROTEINS AND THIS CAN CONFER
GREAT VARIETY OF DIFFERENT
FUNCTIONALITY RANGING FROM CELL
CELL COMMUNICATION,
IMMUNORECOGNITION AND ABERRANT
EXPRESSION OF MANY DISEASE
GLYCAN EXPRESSION IS IMPLICATED
IN A VARIETY OF DISEASE, RANGING
FROM CANCER, CARDIOVASCULAR
DISORDERS AND NEURODEGENERATIVE
DISEASE. CONSEQUENTLY IT IS
IMPORTANT TO QUANTIFY GLYCANS IN
VARIETY OF BIOLOGICAL SYSTEMS.
SO IN OUR RECENT REVIEW ARTICLES
WE HAVE SURVEYED A NUMBER OF
CURRENT STRATEGIES TO QUANTIFY
GLYCOME AND GLYCOPEPTIDES AT THE
FREELY RELEASE GLYCAN LEVEL OR
INTACT GLYCOPEPTIDE LEVEL.
VARIETY OF WAYS TO DO THIS FOR
QUANTITATION LABEL FREE OR
METABOLIC LABELING WITH ISOTOPES
OR ISOTOPES IN CHEMICAL TACT AND
DONE IN MS 1 STAGE, THIS
PARTICULAR TARGET FOCUSES MORE
ON THE MS 2 BASE OR ISOBARRY
TAGGING STRATEGY. THERE ARE
SEVERAL LIMITATIONS WITH CURRENT
STRATEGY. THE METABOLIC LABELING
IS RELATIVELY EXPENSIVE AND ALSO
STABLE ISOTOPE LABELING AT THE
MS 1 STAGE INTRODUCE OVERLAPPING
ISSUE AND ALSO INTRODUCE MORE
CHEMICAL COMPLEXITY AND OFTEN
COME WITH DUPLEX OR TRIPLEX
VERSION. THIS IN PARTICULAR WHEN
INCORPORATING DATA DEPENDENT
ACQUISITION COULD LEAD TO
DECREASED INSTRUMENT DUTY CYCLE
WHICH CREATES SACRIFICE DEPTH OF
QUANTIFICATION AND
IDENTIFICATION. OBVIOUSLY THE MS
2 BASE ISOTAGGING STRATEGY CAN
ADDRESS THESE LIMITATIONS IN ONE
OF THE COMMERCIAL AVAILABLE
PRODUCT AMINOOXI TMT IS
ADDRESSING LIMITATIONS IN TERMS
OF PROVIDING HIGH THROUGH PUT MS
2 BASE QUANTIFICATION. HOWEVER
THERE'S STILL LIMITATIONS THAT I
WILL ELABORATE IN TERMS OF THE
REPORTER AND COMPLEXITY FOR
THESE DIFFERENT GLYCANS SHOWING
HERE IS ISOBARIC LABELING
STRATEGY, COMMERCIALLY AVAILABLE
6 COMPLEX TMT VERSION. WE HAVE
THESE SET UP REPORTER GROUP AND
THEN THE BALANCE GROUP SO SUM OF
THE TWO PORTIONS ARE GIVING RISE
TO IDENTIFY MASS SO ALLOW US TO
DIFFERENTIALLY LABEL FOUR SETS
OF SAMPLES IN THE MS 1 MODE EACH
GLYCAN WILL SHOW AS SINGLE PEAK,
WITH SAME SHIFT AND THEN THE
MSMS WOULD GENERATE THESE
REPORTER ION ON THE PART FROM
EACH OTHER ALLOWING RELATIVE
QUANTIFYCATION. AS WE CAN SEE
THIS IS THE COMMERCIALLY
AVAILABLE TMP REAGENT AND
THERE'S RED STARS INDICATING THE
POSITION OF THE HEAVY STABLE
ISOTOPES. HERE SHOWS THE
ALDEHYDE KETONE REACTIVE GROUP
THAT WILL REACT WITH REDUCING
ENDS OF GLYCANS AND MASS
BALANCER REPORTER. IN THE MSMS
MODE THIS WILL GENERATE THE
CYCLIC REPORTER ION THAT ALLOWS
US TO PERFORM RELATIVE
QUANTIFICATION. HOWEVER THIS
APPROACH SUFFERS FROM SOME OF
THE LIMITATION IN PARTICULAR
LOOKING AT LOW ABUNDANCE COMPLEX
N GLYCANS SO GENERATE IN
PARTICULAR RELATIVELY LOWER
REPORTER ION YIELD AND LEAD TO
INACCURATE REPORTER ION RATIO
AND YOU CAN SEE FROM THIS
EXPERIMENT THE LOW COLLISIONAL
ENERGY WE HAVE MINIMUM
PRODUCTION OF THE REPORTER ION.
SO IN ORDER TO BOOST THAT
REPORTER ION WE NEED TO PERFORM
HIGH ENERGY DISSOCIATION AND
THIS LEADS TO EXCESSIVE
FRAGMENTATION SO NO BACKWARD
FRAGMENTATION DIFFICULTY FOR
IDENTIFICATION. AS A RESULT
OFTEN TIMES WE HAVE TO DO BOTH
LOW ENERGY AND HIGH ENERGY
COLLISION DISSOCIATION. SO TO
ADDRESS THIS LIMITATION OUR
GROUP TALENTED STUDENT HAS TOOK
ADVANTAGE OF NATURALLY OCCURRING
IMMUNOASSAY BUILDING BLOCKS AND
HERE WE SELECTED LEUCINE Z OUR AS OUR
BUILDING BLOCK AFTER
DIMETHYLATION IN PRESENCE OF
GLYCENE METHYL ESTER WE CAN
INSTALL GLYCENE AS LINKER AND
REACT TO FORM THIS HYDROSIDE
REACTIVE GROUP, THIS SUGAR TEST
LABEL ANY CARBONYL CONTAINING
COMPOUND. BY INSTALLING
DIFFERENT STABLE ISOTOPES SUCH
AS COLOR CODED HERE WE CAN
CREATE SHOULDER TAG REPORTER ION
IS ONE APART FROM EACH OTHER
FROM 115 TO 118. WITH THIS SUGAR
LABELING STRATEGY WE DEVELOPED
INTEGRATED WORK FLOW TO LOOK AT
GLYCOME IDENTIFICATION SO FIRST
WE WANT TO EVALUATE WITH
GLYCOPROTEIN BOVINE GLOBULIN
FREELY RELEASED GLOBULIN
DENATURED PENGASE AFTER RELEASE
OF GLYCANS, IN KNOWN RASH YES
AND THEN LABEL DIFFERENTIALLY
LABEL WITH 4 COMPLEX SUGAR AND
MS 1 MODE GENERATE FOR EACH OF
THE SINGLE PEAK AND MS 2 ALLOWS
US TO QUANTIFY. SO THE FIRST
THING WE WANT TO DO IS TO CAM
PAIR THE LABELING EFFICIENCY
COMPARED TO THE COMMERCIAL
PRODUCT. SO HERE WE CAN SEE THAT
THE TOP IS THE UNLABELED NATIVE
GLYCANS AND THE AMINO OXTNT AND
BOTTOM IS SUGAR TAG IN THE
COMMERCIAL PRODUCT WE STILL HAVE
50% UNLABELED PRODUCT AND THEN
WITH OUR SUGAR TAB WE CAN SEE
FOR THESE NUMEROUS DIFFERENT
TYPE OF N GLYCANS WE HAVE 100%
CONVERSION LABELING EFFICIENCY.
ANOTHER VERY IMPORTANT FACTOR TO
COMPARE IS TO FRAGMENTATION
BECAUSE THAT IS WHERE WE BASE
OUR QUANTIFYCATION. SO HERE WE
PERFORM SIDE BY SIDE COMPARISON
WITH THE AMINO OXTNT SUGAR H HCD
COLLISIONAL ENERGY BETWEEN 25
AND 30. IN THIS CASE YOU CAN SEE
THAT IN BOTH CASES SUGAR TAG
PRODUCED MORE INTENSE REPORTER
ION INDICATING THAT WE CAN
ACTUALLY PERFORM RELATIVE
QUANTIFICATION FOR LOWER
ABUNDANCE GLYCANS AN THIS WAS
STILL GENERATE PRETTY ABUNDANT
FRAGMENTATION FOR
IDENTIFICATION. SIMILAR
PERFORMANCE ACHIEVE FOR SIALIC
ACID IN FUCOSE CONTAINING GLYCAN
SHOWN IN THIS EXAMPLE. WE LOOK
AT COLLECTIVE COMPLEX LIKE
PICTURES WITH KNOWN RATIO 1 TO 1
TO 5 TO 10. AS YOU CAN SEE FROM
THIS BOX PLOT WE GET GOOD
QUANTITATION ACCURACY SHOWING
THE BOTTOM ARE TWO
REPRESENTATIVE GLYCAN EXAMPLES,
BOTH FUCOSE AND SIALIC ACID
CONTAINING GLYCANS WE GET PRETTY
GOOD ACCURACY QUANTIFICATION. SO
NEXT WE WANT TO APPLY SUGAR TAG
TO COMPLEX REAL BIOLOGICAL
EXAMPLES SO WE TEAMED WITH CHRIS
IKONOMIDOU AT MEDICAL SCHOOL IN
UW MADISON, HER GROUP IS
INTERESTED IN LOOKING AT THESE
CHEMOTHERAPY TREATED
LYMPHOBLASTIC LEUKEMIA CHILDREN
WHERE WE CAN COLLECT SERUM
SAMPLES AND LOOK AT THE AFTER
CERTAIN TIME PERIOD OF
CHEMOTHERAPY, THE DYNAMIC
CHANGES OF THESE DIFFERENT
GLYCANS. SO HERE SHOWS THE
EXPERIMENTAL SET UPSTART WITH
BASELINE BEFORE TREATMENT AND
ONE MONTH AND THREE MONTHS AND
SIX MONTHS AFTER TREATMENT. SO
THIS WOULD THEN ALLOW US TO
UTILIZE FOUR COMPLEX SUGAR TAG
TO QUANTIFY LARGE NUMBER OF
GLYCANS COLLECTED FROM SERUM
SAMPLES. SO FROM THIS BAR GRAPH
YOU CAN SEE THE Y AXIS IS
TREATED AFTER CONTROL, AND HERE
WE ARE LOOKING AT NUMBER OF
THESE DIFFERENT GLYCANS, ALL
TOGETHER WE WERE ABLE TO
QUANTIFY 145 N GLYCANS INCLUDING
68 N GLYCANS PRESENT IN ALL
THREE PATIENTS AND THESE WERE
DONE WITH THE BIOLOGICAL AND
TECHNICAL TRIPLICATES. SO
OBVIOUSLY THE NEXT STEP FOR US
IS TO FURTHER INCREASE THROUGH
PUT OR MULTIPLEXING CAPACITY
BECAUSE THERE ARE NUMBER OF
ADVANTAGES IN THAT WE CAN REDUCE
AMOUNT OF ANY ONE GIVEN SAMPLE
AND THIS CAN ALSO REDUCE SAMPLE
PREPARATION AND ANALYSIS TIME.
AND MORE IMPORTANTLY, THIS WOULD
ALLOW US TO LOOK AT LARGER
NUMBER OF BIOLOGICAL REPLICATES
OR DIFFERENT CONDITIONS AND
FINALLY THIS COULD IMPROVE LCMS
DATA OVERLAP. SO IN ORDER TO
ACHIEVE THAT, WE HAVE TAKEN
ADVANTAGE OF THE HIGH RESOLUTION
INSTRUMENTATION BUT ALSO
PHENOMENON CALLED MASS DEFECT
AND THE BASIC IDEA IS THAT SMALL
NEUTRON BINDING ENERGY
DIFFERENCE RESULT IN SMALL MASS
DIFFERENCE, IF WE SWAP WITH
CARBON 13 THIS REEDS TO 2.9
MILIDALTON MASS DIFFERENCE, NOT
DIFFERENTIATED WITH HIGH
RESOLUTION MASS SPEC
INSTRUMENTATION. SO HERE ARE
BASED ON THIS IDEA WE CAN TRIPLE
THE THROUGH PUT OF ORIGINAL FOUR
COMPLEX SUGAR TAG TO GENERATE 12
TAG SUGAR TAG AND HERE SHOWS THE
RESOLVING POWER, 30,000
RESOLVING POWER WE CAN MICELY
RESOLVE THESE 12 QUANTIFICATION
CHANNEL. SO HERE SHOWS CENSUS,
WE CAN -- THIS WAS DONE WITH
GRAD SUNT IN THE LACK MIC LI. SO
WE CAN USE THE -- IN THE LAB
MIKE LI SO WE CAN GENERATE 12
COMPLEX SUGAR TAG. IF WE SWAP
CARBON 13 THERE IS A 6
MILIDALTON MASS DIFFERENCE
DIFFERENTIATED BUT IN LOW
RESOLUTION THIS MAY SEEM AS A
SINGLE PEAK, THE 115A ANDB
HOWEVER WAS HIGH RESOLUTION, WE
CAN RESOLVE THEM SO THIS IS
ACTUALLY SHOWING THE REQUIRED
MASS SPECTRAL RESOLUTION THAT
YOU CAN SYSTEMATICALLY RESOLVE
BASELINE RESOLVE THESE 12
REPORTER IONS YOU CAN SEE AGAIN
AT 30,000 OR EVEN 60,000 WE CAN
NICELY TELL THESE 12 CHANNELS
APART FROM EACH OTHER. HERE WE
ARE AGAIN TAKING FREELY RELEASED
BTG GLYCANS AND LOOK AT KNOWN
RATIO WITH THIS TALL PLEX SUGAR
TAG AND ON THE LOWER PANEL IS
EXAMPLE OF NUMBER OF GLYCANS IN
MSMS AND ALSO ZOOM IN FOR THE
QUANTIFICATION THAT ALLOWS US TO
QUANTIFY THESE THINGS. SO IN
THE REMAINING TIME I WANT TO
ALSO TELL YOU A LITTLE BIT ABOUT
BESIDES LOOKING AT FREELY
RELEASE GLYCANS WE CAN ALSO LOOK
AT INTACT GLYCOPEPTIDES IN
PARTICULAR THE GLYCOFORM BECAUSE
THAT'S ALSO IMPLICATED IN
DIFFERENT DISEASE CONDITIONS.
HERE WE TEAMED UP WITH WISCONSIN
ALZHEIMER'S DISEASE RESEARCH
CENTER AND WE DEVELOPED THIS
INTEGRATED ANALYTICAL WORK FLOW
TO LOOK AT GLYCOPROTEOME FROM
CEREBRAL SPINAL FLUID COLLECTED
AT DIFFERENT DISEASE STAGE AND
AFTER PROTEIN EXTRACTION
DIGESTION AND ENRICHMENT AND
ALSO WE HEARD ABOUT
FRAGMENTATION TECHNIQUE FOR
INTACT GLYCOPEPTIDE
QUANTIFICATION. HERE WE USING A
DIMANY METHYLATED LEUCINE TAG TO
QUANTIFY THE GLYCOPEP SIDES.
HERE IS GENERIC STRUCTURE IS A
MEAN REACTIVE GROUP WITH BALANCE
CARBONIL GROUP AND DIMETHYLATED
LEUCINE WITH STABLE ISOTOPES SO
HERE IS DUPLEX VERSION AND HERE
USING THE SAME HIGH RESOLUTION
MASS DEFECT PHENOMENON WE CAN
CREATE A 12 PLEX CHANNEL. THIS
12 PLEX WE CAN INTERROGATE 48
PATIENTS, DIVIDE INTO LEE
GROUPS, CONTROL MCI MILD
COGNITIVE IMPAIRMENT AND ADVANCE
DEMENTIA, 16 PATIENT EACH AND
WITH THIS INTEGRATED WORK FLOW
WE 12 MENTION QUANTIFY
GLYCOPEPTIDES TO 107 IN
GLYCOPROTEINS.
. WE LOOK AT THREE OF THESE
GLYCOPROTEINS RELATED TO AD. THE
GLYCANNER -- THE NEURAL CELL
ADHESION MOLECULE, SO THIS ONE
HAS FOUR DIFFERENT GLYCOSIDE AND
THERE IS MINIMUM DEGREE OF
GLYCAN HETEROGENEITY, COMPLIMENT
CS 3 IS NEURAL INFLAMMATORY
PROTEIN HAS SINGLE SITE ENCODE
22 DIFFERENT GLYCOFORMS. PERHAPS
MOST INTERESTING IS CLUSTERING
AND THIS ONE IS THE THIRD MOST
RISK GENE IN AD, AND ENCODES 449
AMINO ACID RESIDUE, FOUR
GLYCOSIDES AND SOME OF THESE
MINIMUM DEGREE OF GLYCAN
HETEROGENEITY BUT IMPORTANTLY
THIS 374 ENCODES 46 -- 56
GLYCOFORMS MANY HAVE SIALIC ACID
AND FUCOSYLATION. SO WE HAVE
ALSO LOOK AT GLOBALLY THESE
DISREGULATION OF M GLYCOPEPTIDES
IN THIS PANEL PLOT SHOWED AT THE
NCI AND NUMBER OF N GLYCOSIDES
AND GENE ONTOLOGY ANALYSIS
REVIEWED A GLYCOPROCESS
INCLUDING PLATELET DEGRANULATION
CELL ADHESION AND INFLAMMATORY
RESPONSE RELATED TO THE
MOLECULAR DEGENERATION. SO THEN
WE HAVE LOOK AT SOME OF THESE
GLYCOPEPTIDES AT NCI AND AD AND
THIS REALLY SHOWED NOT ONLY
REALLY THE SIGNIFICANT CHANGES
OCCURRING AT THE GLYCOSIDE, N
GLYCOPEPTIDES, NOT AT PROTEIN
LEVEL. SINCE I'M RUNNING SHORT
ON TIME I'M GOING TO CLOSE AND
TO POINT OUT THAT WE DIVIDE
DISEASE STAGE SPECIFIC IN
GLYCOPEPTIDES. SO TO WRAP UP I
HAVE SHOWN YOU BEDEVELOPED SET
OF GENERIC LABELING REAGENT THAT
ALLOWS US TO PERFORM HIGH
THROUGH PUT GLYCAN
QUANTIFICATION AND GLYCOPEPTIDE
QUANTIFYCATION. AND SO NUMBER
OF PUBLICATIONS ACKNOWLEDGING
THE UO 1 GRANT ALL TOGETHER, WE
IN THE PAST THREE YEARS
PUBLISHED 30 PUBLICATIONS AND
NUMBER OF THESE TALKS AND
SESSIONS IN PARTICULAR, THIS
UPCOMING JUNE WE HAVE THE NORTH
AMERICAN MASS SPEC SUMMER
SCHOOL, TALK MORE ABOUT OUR
TECHNOLOGY AND GREAT VARIETY OF
DIFFERENT COLLABORATIONINGS.
WITH THAT I WANT TO THANK YOU
ALL FOR KIND ATTENTION AND ALSO
AGAIN THANK YOU TO GLYCOCOMMON
FUNDS AND ALL OF YOU FOR YOUR
KIND ATTENTION. THANK YOU.
[APPLAUSE]
>> OUR NEXT SPEAKER IS
(OFF MIC)
LORI YANG.
(OFF MIC)
>> CAN EVERYONE HEAR ME? GREAT.
SO I WANT TO REITERATE OUR DEEP
GRATITUDE TOWARDS THE NIH AND
COMMON FUND OFFICIALS FOR ALL
THEIR SUPPORT THROUGHOUT THE
YEARS AND FOR MAKING THIS FINAL
MEETING HAPPEN AND IN PERSON.
FIRST LET ME TELL YOU A LITTLE
BIT ABOUT OUR COMPANY. WE ARE A
FOR PROFIT COMPANY. WE ARE AN
ACADEMIC SPIN OFF OF THE LAB OF
PROFESSOR ROB WOODS CO-FOUNDER
AND PRESIDENT OF LECTENZ BICO
YOU HEARD FROM EARLIER AND WILL
HEAR FROM AGAIN LATER. WE ARE
HEAD QUARTERED AT THE INNOVATION
GATEWAY INCUBATOR AT UNIVERSITY
OF GEORGIA. WE HAVE A SATELLITE
FACILITY IN SOUT SAN DIEGO
CALIFORNIA. SO WHAT IS
GLYCOSENSE? A SOLUBLE LECTIN
MICROARRAY BUT INSTEAD OF THE
LEPTONS BEING SPOTTED ON TO A
MICROSCOPE SLIDE, THEY ARE
CHEMICALLY CONJUGATED TO
MICROSPHERES AND SUSPENDED IN
SOLUTION. SO IT IS A MIX AND RUN
ASSAY.ASSAY. GLYCOSENSE BEADS ARE
MULTIPLEX MICROSPEARS WITH
DISCREET LEVELS OF RED
FLUORESCENCE INTENSITY AND EACH
IS CONJUGATED TO A DIFFERENT
GLYCAN BINDING PROTEIN. THE
BEADS ARE MIXED WITH FLUORESCENT
GLYCOPROTEIN ANOLYTE EITHER
DIRECTLY LABELED OR SUBSEQUENTLY
INCUBATED WITH SECONDARY
REAGENT. AS SHOWN HERE. AND THE
BEADS AND SAMPLE ARE THEN RUN ON
A FLOW SIGH TOM TEAR. HYDRO
DYNAMICALLY FOCUSES BEAD SUCH
THAT THEY RUN SINGLE FILE
THROUGH THIS ENLARGED FLOW CELL.
A LASER INTERROGATES EACH BEAD
BECAUSE IT IS JUST THE BEAD AND
NOT SURROUNDING SOLUTION, WASHES
ARE USUALLY UNNECESSARY. SO LET
ME TELL YOU A LITTLE BIT IN
DETAIL HOW WE GET FROM THE DATA
TO THE FINAL GLYCOPRINT. FIRST
LIGHT SCATTER IS MEASURED. FORD
AND SIDE SCANNER SORT OF RELATE
TO THE SIZE AND GRANULARITY OF A
CELL. BUT IN OUR CASE WE ARE
LOOKING AT BEADS. SO EACH DOT
REPRESENT AS BEAD. BECAUSE BEADS
ARE SO UNIFORM THEY CLUSTER
TIGHTLY AROUND THE CENTER. SO WE
DRAW A GATE TO ANALYZE SINGLE
BEAD AND NOT DUB BEADS OR
DEBRIS. THEN THE RED
FLUORESCENCE INTENSITY OF EACH
BEAD IS NEGATIVE. AS I SAID
BEFORE EACH BEAD SET HAS A
DEFINED AMOUNT OF RED DYE
INCORPORATED SHOWN SCHEMATICALLY
HERE AS A DIFFERENT LEVEL OF RED
COLOR. AND IT IS FIVE CP. SO BP
IS GATED, SO THAT THE GREEN
FLUORESCENCE OF EACH RED
FLUORESCENT BEAD CAN BE
MEASURED. AND WE SEE THAT ONLY
THE GAL AND GLCNAC DETECTING
BEADS HAVE A HIGH LEVEL OF GREEN
FLUORESCENCE IN THE X AXIS. AND
THAT'S BECAUSE THEY HAVE PROTEIN
BOUND ENLARGED ON THE RIGHT IT
HAS ONLY TERMINAL GAL AND
GLCNAC. TO SHOW ALL THE DATA ALL
TOGETHER HERE IS ANOTHER
BIVARIANT DOT PLOT, AGAIN, EACH
DOT REPRESENTS THE BEAD SO THE
BEADS ARE DISTINGUISHED BY THEIR
POSITION ALONG THE X AXIS WHILE
ANY GREEN FLUORESCENT
GLYCOPROTEIN BAND IS OBSERVED ON
THE Y AXIS AT THIS TIME AND ONLY
THE GAL AND GLCNAC HAVE
DETECTING BEADS AND NOT THE
OTHERS. SO THIS COB CONVERTED
INTO -- CONVERTED TO A SIMPLE
GLYCOPRINT, BAR GRAPH OF THE
GREEN FLUORESCENCE INTENSITY OF
EACH GLYCAN DETECTING BEAD. JUST
TO NOTE, THE RATIO IS
PURPOSEFULLY NOT ONE TO ONE
BECAUSE THIS IS A NOT AN
ABSOLUTELY QUANTITATIVE ASSAY,
EACH GLYCAN DETECTING REAGENT
HAS DIFFERENT SPECIFICITIES FOR
GLYCAN SO THIS ACTION IS BEST
FOR LOOKING AT CHANGES. ANOTHER
NOTE, IN REALITY THAT WAS A
SIMPLE UNIFORMLY GLYCOSYLATED
GLYCOPROTEIN. BUT IN ACTUALITY
GLYCOPROTEINS HAVE MULTIPLE
SITES OF GLYCOSYLATION AND
DIFFERENT GLYCOFORMS AT EACH
SITE. SO WHAT WE ARE LOOKING AT
IS A REPRESENTATION OF THE
MIXTURE OF GLYCOFORMS AND SENSE
N GLYCANS SHARE COMMON CORE WE
ARE ONLY INTERESTED MANY THE
CAPCAP. WE CHOSE FCSs PRESS TO
ANALYZE THE DATA BECAUSE THEY
OFFER A FREE VERSION FOR WHICH
WE CREATED THEM PLACE WITH
INTEGRATED SOPs SO THAT USERS
COULD UPLOAD THEIR DATA FILES
AND FOLLOW THE STEP BY STEP
INSTRUCTIONS FOR ANALYSIS BUT
USERS HAVEN'T REQUESTED OR
NEEDED THIS BECAUSE IT IS EASY
ENOUGH TO TAKE THE DATA OFF THE
INSTRUMENT. YOU CROSS VALIDATED
ASSAY ON OTHER SIGH TOM TEARS
USING CONTROLLED GLYCOPROTEINS
FLUORESCFLUORESCENT -- ALL THESE
INSTRUMENTS GAVE THE SAME DATA
EXCEPT THE NEXT WHICH IS AN
IMAGING CELL COUNTER. WE CROSS
COLLABORATED WITH COLLABORATORS
BY SENDING THEM KITS AND
FORTUNATELY THE CONTROL
EXPERIMENTS WORKED ON THEIR
INSTRUMENTS IN THEIR HANDS. AND
SO MAKE SURE WHAT REALLY CROSS
VALIDATED BY OTHER METHODS, TOP
ROW OR OUR GLYCOPRINTS. WE TOOK
FLUORESCENTLY LABELED FETERIN, A
HIGH LEVEL SIALIC ALPHA 26 AND
ALPHA 23 WITH SIALIDASE AND WE
SAW THAT THE SIALIDASE SIALIC
ACID LEVELS DECREASED WHILE THE
GALACTOSE LEVELS INCREASED AND
UPON ADDITION OF GAY LACTOSEISIT
THE GALACTOSE SIGNALS DECREASED
WHILE GLCNAC INCREASED FROM
BEING EXPOSED. CONSISTENT WITH 2
AB PROFILING IN MASS SPEC, THOSE
ARE THE GLYCOFORMS THAT ARE SEEN
IN ALL THREE POINTS. SO HERE
JUST A FEW APPLICATIONS OF
GLYCOSENSE YOU CAN DO SIMPLE
GLYCOPROTEIN ANALYSIS AND
GENERATE THESE GLYCOPRINTS, YOU
CAN MONITOR GLYCOPROTEIN
PRODUCTION IN PROCESS. AND YOU
CAN MONITOR GLYCOENGINEERING OF
GLYCOPROTEINS THAT IS
GLYCOSIDASE AND GLYCOSYL
TRANSFERASE REACTION. YOU CAN
ALSO LOOK AT THE GLYCOSYLATION
OF NATIVE ANTIBODIES. SO WE
HAVE USED FLUORESCENTLY LABELED
GLYCOPROTEINS FOR SIMPLICITY TO
HELP DEVELOP METHODS, ESPECIALLY
FETUIN BUT THIS WORKS WITH
INDIRECT DETECTION AS WELL. IF
WE TAKE UNLABELED FETUIN AND
INCUBATE WITH RB AND FOLLOWED BY
LABELED ANTI-FETUIN FAB THAT
FRAGMENT BECOMES ANTIBODIES
THEMSELVESES ARE GLYCOSYLATE WED
SEE THEY GIVE SIMILAR
GLYCOPRINTS. WE USE GLYCOMONITOR
GLYCOPRODUCTION PROTEIN IN
PROCESS, IN THIS CASE ROBO 1
GLYCOPROTEIN WHICH HAS A SINGLE
SITE N GLYCOSYLATION AND IS
CONVENIENTLY GFP TAGGED SO THAT
THE FLUORESCENT READ OUT IS
BUILT RIGHT IN. WE INCUBATED
GLYCOSENSE BEADS WITH CELL
CALLTURE MAINTENANCE, WE DIDN'T
PRE-PURIFY OR DO WASHINGS OR
RELEASE GLYCANS OR ANYTHING LIKE
THAT. ON THE LEFT THEY ARE ROBO
1 GLYCOPRINTS EACH DAY OVER
SEVEN DAYS OF PRODUCTION AND TO
LOOK AT IT ANOTHER WAY, ON THE
RIGHT IS A SIMPLE LINE GRAPH OF
EACH GLYCOFEATURE MONITORED OVER
IN A TIME COURSE. AND NORMALIZE
TO TOTAL PROTEIN AMOUNT. THANKS
TO KELLY MOREMEN FOR THE ROBO 1
AND PARASTOO AZADI FOR THE MASS
SPEC DATA. CONSISTENT WITH MASS
SPEC THERE IS A HIGH PERCENTAGE
OF TERMINALLY GLYCOSYLATED AND
FEWS TO LATED GLYCOPERFORMS AND
LOW PERCENTAGE OF U -- WE USE
GLYCOSENSE TO MONITOR
GLYCOENGINEERING. AND THIS IS
FETEUIN ANOTHER TIME COURSE
TREAT WITH SIALIDASE FOR ONE
HOUR FOLLOWED BY GALACTOSIGH
DAYS FOR TWO HOURS. YOU SEE THE
BEGINNING AND END POINTS OF THE
REACTION BUT HERE WE ARE
ACTUALLY FOLLOWING THE REACTION
AT 15 MINUTE INTERVALS SO YOU
CAN SEE THE SIALIC ACID SINGLE
PURPLE LINE DECREASE WITH
ADDITION OF SIALIDASE WHILE
GALACTOSE SIGNAL INCREASES. AND
THE YELLOW LINE AND UPON
ADDITION OF GALACTOSIDASE THE
GAL SIGNAL DECREASES WHILE
SIMULTANEOUSLY THE GLIK MA'AM
SIGNAL ON THE OPPOSITE LINE. SO
WE PUT GAL BACK ON OR SIALIC
ACID BACK ON SO ON THE LEFT SIDE
THE BEADS ARE SHOWN SIDE BY SIDE
TO SHOW THE DIFFERENCE BETWEEN
THE ENZYME AND UNTREATED AND
TREATED GLYCOPROTEIN. SO
AGALACTOASIALFET ORBSIN BLUE BAR
HAS HIGH LEVEL OF GLCNAC BUT
SIALYLTRANSFERASE THE GAL GOES
UP AND GOES DOWN TREATED BY GAL.
WE USED ALPHA 26 OR ALPHA 23 AND
WE SEE UPON ADDITION WITH THE 26
THAT THE 26 SIGNAL GOES UP, THE
ORANGE BAR, NOT THE 23 AND THE
GAL SIGNAL GOES DOWN. SIMILARLY
UPON TREATMENT WITH 23
SIALYLTRANSFERASE THE 2, 3 GRAY
BAR GOES UP, NOT 26 AND THE GAL
GOES DOWN. SO AGAIN NOT
QUANTITATIVE, NOT A ONE TO ONE
RELATIONSHIP. WE ALSO LOOKED AT
NATIVE ANTIBODY WHICH IS IS
DIFFICULT BECAUSE THEY DON'T
LOOK LIKE THIS, THEY LOOK LIKE
THIS. THE GLYCANS ARE SOMETIMES
INACCESSIBLE AND DIFFERENT
ANTIBODIES SUB CLASSES REACT
DIFFERENTLY IN OUR PHASE WE HAVE
TO TEST ON A CASE-BY-CASE BASIS.
SO IN THIS EXAMPLE HERE IS
PROTEIN G PURIFIED HUMAN IGG AND
TO MAKE SURE WHAT WE ARE SEEING
IS REAL, WE TOOK THE DIRECTLY
LABELED IGG IN BLUE, TREAT WITH
SIALIDASE, THE SIALIC ACID
SIGNAL GOES DOWN AND GAL SIGNAL
GOES UP AND UPON TREATMENT WITH
GALACTOSIGH DAYS THE GAL GOES
DOWN AND GLCNAC GOES BACK UP.
THANKS TO -- FOR THE SAMPLES AND
SHE RECEIVED AN ADMINISTRATIVE
SUPPLEMENT TO DO HIGH THROUGH
PUT ANALYSIS OF SIALYLATION OF
IGG FROM HUMAN AND MOUSE MODEL.
SO IN SUMMARY, IT IS AND IN
PROCESS GLYCO ANALYSIS METHOD
THAT CHARACTERIZES IT IS
MULTIPLEX RUN ALL THE BEADS AT
ONCE, MIX AND MATCH CHOOSE LEVEL
FEATURES YOU WANT TO DETECT, NO
NEED FOR RELEASE OF GLYCANS FROM
WASHING, VERY EASY TO USE. DON'T
NEED HIGH AMOUNT OF TRAINING,
SPECIAL EQUIPMENT OR SOFTWARE
JUST BASIC FLOW SIGH CYTOMETER.
AND FAST, CAN BE MONITORED IN
NEAR REAL TIME AFTER SHORT
BINDING STEP. WE DID LAUNCH THIS
PRODUCT IN IN JUNE OF 2020 AS
THE DIFFICULT TIME BUT IT
INCLUDES THE FOLLOWING BEADS,
BACKGROUND OF 23 GAL GLCNAC
DETECTING BEADS AND TUBE OF
GATING BEADS, THE FIVE
UNCONJUGATED BEADS. GLYCOPROTEIN
CONTROLS. AND THIS IS AVAILABLE
ON OUR WEB SHOP ALSO ON FISHER
SCIENTIFIC, YOU HAVE TO SEARCH
FOR THE PRODUCT AND FOR ANY UGA
PEOPLE LISTENING WE ARE ALL OUR
PRODUCTS ARE ON UGA MART AND FOR
INSTITUTIONS THAT USE THE JAGGER
PLATFORM ALL OUR PRODUCTS ARE ON
THERE. FINALLY I WOULD LIKE TO
THANK EVERYBODY PAST AND PRESENT
ON THE LECTENZ BIO TEAM, MATT
WHO DEVELOPED AND PERSONOR
COMMERCIALIZED THE KIT HE'S
MOVED ON BUT MELINA HAS
ADMIRALLY TAKEN UP PRODUCTION
AND OUR FUNDING SOURCES,
ESPECIALLY COMMON FUND TO
DEVELOP THIS PLATFORM AND
COMPANION SBIR TO DEVELOP THE
GLYCOENGINEERING APPLICATION.
ALL OF OUR BETA TESTERS
COLLABORATORS CUSTOMERS, CAN SEE
OUR PRODUCTS ON THE WEBSITE AND
FOR COLLABORATIONS OR QUESTIONS
PLEASE CONTACT ME DIRECTLY OR
VISIT US AT OUR POSTERS OUTSIDE.
THANKS VERY MUCH FOR YOUR
ATTENTION.
[APPLAUSE]
THANK YOU, PAM FOR LEADING SO
WELL AND ORGANIZING THIS MEETING
AND BY THE WAY HOW WAS DINNER
LAST NIGHT? WHAT ARE LECTENZ,
NORMAL CLASS OF TARGETING
REAGENTS THAT ARE LECTIN LIKE
AND ENZYME DERIVED. MADE FROM
ENZYME BECAUSE THEY ARE MADE
FROM ENZYME THEY ARE DIFFERENT
FROM NATURAL LECTENZ IN MANY
WAYS AND THEY HAVE TUNEABLE
AFFINITIES IN BINDING KINETICS.
AND THOSE ARE ESSENTIAL FOR
AFFINITY OF PUBLICATION, ON THE
ENZYME PROPERTY AND THEY ARE
MONOMERIC AND THEY ARE
MONOMERIC, MINIMIZE THE --
THROUGH CELLTURE CELLS, THAT'S
GOOD FOR STAINING OF LIVE CELLS.
THEY ARE COMMONLY EXPRESSED IN
E. COLI SO PRODUCE LARGE AMOUNT
AND MINIMIZING -- DURATION AND
MOST IMPORTANTLY IN THEORY,
EVERY SINGLE CARBOHYDRATE
PROCESS CAN BE CONVERTED INTO
LECTENZ. HOW THEY ARE MADE.
SIMPLY THEY ARE COMPUTATIONAL
GUIDED DIRECTLY EVOLUTION. THEY
ARE MADE AND START WITH CAUSE OF
ENZYME WITH KNOWN STRUCTURE, WE
ALL KNOW THAT, AND USE THE
COMPUTATIONAL MODEL TO IDENTIFY
SITES TO MUTATE AND WE USE THOSE
DATA MODERN DATA TO DESIGN
COMMON TORRIAL LIBRARY AND
EXPRESS -- COMBINATORIAL LIBRARY
AND OTHER RUNGS OF MY OPINION
AND WE IDENTIFY THOSE HIGH
BINDERS, EXPRESS, WE TAKE THOSE
HIGH BINDERS, GROUND THEM IN E.
COLI AND HAVE THE PROTEIN
VALIDATED FOR BINDING AFFINITY
AND BINDING SPECIFICITY. OF
COURSE WE WANT TO PUT THOSE
TOIUS BY BETA TESTING
APPLICATIONS. MODERATE IS KEY
TO LECTENZ DEVELOPMENT. THIS IS
JUST ONE OF EXAMPLE, COMPLEX
CRYSTAL STRUCTURE WITH LIGAND
AND IF NOT THERE IS -- SOME OF
THEM WILL BE RUN THREE OR FOUR
WEEKSWEEKS. COME UP WITH THREE GROUPS
OF RESIDUES AND RED ONE IS THE
ONE THAT FAVORABLE RESIDUE THOSE
ARE INVOLVED IN ENZYME ACTIVITY
WE DON'T NEED SO WE WANT TO
MUTATE THOSE. AND ANOTHER GROUP
IS THE BLUE ONE WE IDENTIFY,
THOSE ARE CALLED TEPID RESIDUES.
SO CLOSE TO LIGAND BUT UNDER
5-ANGSTROM OR LESS. BUT THEY
BARELY CONTRIBUTE ENERGY TO THE
BINDING. SO THOSE ARE THE ONE WE
MUTATE ONE BY ONE EACH O OTHER
ASSAYS AND EVERYTHING COMBINED
TO MAKE LIBRARY. THIS IS JUST
ONE EXAMPLE OF WE START WITH
ENGINEER SIALIC ACID LECTINS BY
ENDURING FROM METHYL SAL DAYS B,
CAME ONE RUNGS AFTER GOING
THROUGH PROCESS AND WE CAME UP
WITH TWO LECTINS ONE WE CALL
TWO, THREE SPECIFIC, OTHER IS
PAIN SPECIFICKER 5 GC AND
LINKAGE INDEPENDENT MANNER, IT
WILL BIND TO OTHER 2, 3, HOUR 23
AND HOUR 28. THIS SHOWS HOW WE
MADE THE BINDING CONSTANT, THIS
IS BY MEASUREMENT AND EVERYTHING
IS ONE MICROMOLAR OR SMALLER,
BINDING CONSTANT AND TARGET OF
LEPTONS. THIS IS WE WANT TO SEE
HOW SPECIFIC REAGENTS ARE, ONE
BINDING ARRAY, IN THE COLUMN IN
THE LAB IN UNIVERSITY AND GOING
TO SHOW THE DATA FROM HER LAB
BUT WE ALSO HAD GET SURVEYS FROM
NCFG AND Z BIOTECH AND THEY
PRODUCE HAVE SIMILAR RESULTS.
WE CAN SEE THAT, SPECIFICALLY
BIND TO OTHER 23, 26, 28 AND
IDENTIFY A 5 AC SO TWO DAY PAN
SPECIFIC. ONE THIS RUNS ONE DOWN
SIDE OF THIS REAGENT IS THAT IT
IS NOT REALLY (INAUDIBLE)
SENSITIVE O -- WE HAD TO DO
BINDING DETECTION IN THE VERY
LOW SAM, INCREASE SALT SIGNAL
WOULD BE REDUCED. SO WE WENT TO
DO ANOTHER RUNS OF MUTAGENESIS
AND THIS COME UP WITH VERSION 2,
SO THIS IS VERSION 2 OF THESE
PAN SPECIFIC NOW SIGNAL HAS BEEN
INCREASED ABOUT 200 TIMES. LESS
SENSITIVITY. SO IT IS ON THE --
THESE SHOW YOU THE SPECIFICITY
IS THE SAME, STILL SPECIFIC. 23
SPECIFIC. HOWEVER WHEN THESE
LEVELS AMONG OTHER THINGS.
COUPLE OF EXAMPLES WITH WESTERN
BLOT, VERY SPECIFIC 23, HOUR 23
AND PAN SPECIFIC AND THESE ALPHA
26 IS NOT LECTENZ, IT IS
ENGINEERED FROM MUCH ROOM
LEPTON. COMPLETE SPECIFICITY.
THIS IS BRIGHT ONE IHC AND DATA
ALPHA 23 SPECIFIC BINDS MORE
QUITE SEEMS TO FOCUS ON THESE
VARIOUS CELLS AND THESE OTHER
TWO IS CONFINED MOSTLY TO THIS
LAMINA PROPRIA. AND IF WE TREAT
WITH NEUROLAMIDASE THE SIGNAL IS
GONE FORMAL 23 AND ALPHA 26 IF
YOU STAIN WITH ECA AFTER ALPHA
23 SIGNAL INCREASE, BECAUSE YOU
GET RID OF SIALIC ACID AND THE
-- GET EXPOSED SO YOU SEE MUCH
MOVE SIGNAL ESPECIALLY IN THE
AREAS YOU SEE SIGNAL ECA, OVER
THE UNTREATED. SIGNAL THERE.
THIS IS RESULT FROM COMMON
CENTER. AND ON THIS PICTURE YOU
-- THOSE ARE SKIN CELLS AND
SIALYLTRANSFERASE, IT IS GONE.
SO IN THE CASE OF STOICHIOMETRY
IT IS CLEAR WE TREAT WITH
NEURAMINIDASE, A SIGNAL IS GONE.
IT BECOMES RED ONE MEANS THERE
IS NO BINDING. THIS IS ON THE
LIVE CELLS CULTURE CELLS. OKAY.
THE APPLICATION CAN ALSO USE
AFFINITY PURIFICATION AND THIS
IS ONE WE MIX EQUAL AMOUNT OF
HOUR 23 AND ALPHA 26
GLYCOPROTEINS AND THE WESTERN
BLOT SHOW YOU THERE IS A CLEAR
SEPARATION HERE OF THESE TWO. SO
ALPHA 23, THIS WILL BE ALPHA 2,
6, THIS IS ALPHA 2, 3 AND
WESTERN BLOT SHOWS RESULT IN
HERE. WE ALSO USE OUR REAGENTS.
THIS IS ANOTHER EXAMPLE WE USE
NOVEL ONE EXPRESS HK 293 CELLS
PROVIDED BY (INAUDIBLE) YOU CAN
SEE THAT WE CAN HAVE THE PROTEIN
PURIFIED, COME OUT WITH ABOUT
20% OF TOTAL AMOUNT OF EXPRESS
AND WESTERN BLOT SHOWS THAT IN
THE FLOW THROUGH IN THE FLOW
THROUGH YOU SEE ILLUSION WITH
ALPHA 2, 3 SPECIFIC PROBE IS
USED IT IS USED YOU SEE MOST OF
THEM SIGNAL COMES BECOMES AN
ILLUSION A LITTLE BIT MANY THE IN THE
INPUT IF YOU USE ALPHA 26 PROBE
YOU SEE FLOW THREE MOSTLY IS
ALPHA 2, 6 BUT ALSO DETECT ALPHA
2 ON THE ILLUSION AS WELL THIS
IS BECAUSE FRACTION CONTAINS
THOSE ALPHA 2, 3 AND ALPHA 2, 6
IN SINGLE. (INAUDIBLE) LAB DID
SERVICE FOR US AND YOU CAN SEE
THAT THERE IS A CLEAR ENRICHMENT
OF SIALYLATION ENRICHMENT.
BECAUSE WE HAD TO MAKE ENZYME
FROM THERE, WE HAD TO START WITH
ENZYME TO MAKE THE LECTENZ SO
EXPRESS IN WAY HIGH AUTHORITY
WITH LOW COST SO WE DECIDED TO
MAKE THOSE LEVELS OF
GLYCOSCIENCE COMMUNITY AND THAT
HE ARE ON OUR WEBSITE. THANK
YOU TO THE GROUP. THIS IS OUR
GROUP, I WANT TO THANK THE PAST
MEMBERS AND OUR COLLABORATOR AND
SERVICE PROVIDERS. THANK YOU
VERY MUCH.
[APPLAUSE]
>>STUDENT: SOFT (INAUDIBLE) THE
LAST TALK.
>> 3-D STRUCTURE. WHAT YOU SEE
IS AN MD SIMULATION OF CAPSID
POLYSACCHARIDE FRAGMENT AND WHAT
IT REALLY POINTS OUT AL HOE WE
AS GLYCOBIOLOGISTS SAY
CARBOHYDRATE ARE FLEXIBLE IT IS
NOT ALWAYS CLEAR THAT WE ARE
TRULY HAVE THAT IN OUR HEADS.
BECAUSE WE WILL SAY AND I GET
ASKED THIS ALL THE TIME, WHAT IS
THE SHAPE OF MY SUGAR? AND I
WILL SAY IT IS FLEXIBLE, I KNOW
WHAT IS ITS SHAPE? SO YOU HAVE
TO THINK OF SHAPE AS AN ENSEMBLE
OF PROBABLE SHAPES. THE MOST
COMMON SHAPE AND OTHER SHAPES.
ONE OF THE CHALLENGES WE FACE IS
HOW DO YOU REPRESENT IN A VIDEO
IT IS GREAT BUT YOU DON'T
PUBLISH A VIDEO. YOU NEED SOME
OTHER WAY. WHAT YOU SEE RIGHT
SIDE IS A MOSS PLAT PLOT BECAUSE
SUGARS ON SURFACE LOOK LIKE
MOSS, THIS IS AN OVERLAY OF
MULTIPLE SHAPES FROM AN MD
SIMULATION. SO JUST A WAY TO
REPRESENT THINGS BUT THIS IS A
REALM OF SPECIALIST AND OUR
WHOLE GOAL OF THIS TOOL WE HAVE
DEVELOPED AND ARE CONTINUING TO
DEVELOP IS TO GET IN THE HANDS
OF THE NON-SPECIALISTS. SO HERE
IS OUR WEBSITE I POINTED OUT
EARLIER PDB SEARCH, WHAT I WILL
TALK TO YOU ABOUT NOW IS HOW DO
YOU MODEL THE SHAPES OF SUGAR.
THREE WAYS TO DO THIS ONE IS THE
POINT AND CLICK THAT WE SHOWED
YOU GRAPHICAL USER ENTERPACE AND
THAT IS THE WAY I MOST COMMONLY
DO IT BUT THERE ARE OTHERS AND
ONE WAS EARLY ADOPTED BY CFG
WHICH IS PASSING THROUGH THE URL
STREAM SO WHEN YOU RUN HTTP BLAH
BLAH BLAH SLASH DA, DA, DA.
THERE IS A WAY THE DO THAT AND
YOU CAN ENCODE IT AND IT WILL
SENSE SEQUENCE OF SUGAR TO OUR
SITE AND BUILD IT. THERE IS A
MORE SOPHISTICATED WAY TO ENCODE
MORE INFORMATION THAN API. THIS
IS ADOPTED BY GLYCAN THAT ALLOWS
REMOTE WEB SITES OR DATABASES TO
GENERATE 3-D STRUCTURE
AUTONOMOUSLY. IN TERMS OF
USAGE, THE TOOLS HAVE BEEN VERY
WIDELY USED. IF WE LOOK BACK SAY
2014 WE HAD MAYBE 20 PAPERS
SITING EITHER TERM GLYCAN WEB OR
TERM GLYCAN.ORG. NOW WE ARE ON
PROJECTED TO HIT ABOUT 100
PAPERS. OF THAT COUPLE ARE MINE.
I HAVEN'T FILTERED THIS OUT BUT
CERTAINLY NOT THAT MANY. YOU
HAVE SEEN THAT ABOUT THE SAME
AMOUNT MENTION GLEE CAM WEB OR
GLEE CAM.ORG. THE ONES SAYING
WEB ALSO AGREE CAM.ORG. BUT THEY
MAY OR MAY NOT SAY THE WORDS
GLYCAM WEB SO PROBABLY MORE THAN
A HUP PAPERS A YEAR COMING OUT
NOW. WHAT IS MUCH MORE
INTERESTING THOUGH IS ARE THE
SAY THE USERS OF THIS GOOI, IF
WE LOOK AT VISITORS WE ARE UP
AROUND 50,000 A YEAR. WHAT
CALLED UNIQUE VISITS. OR
VISITORS. ABOUT TWICE THAT ARE
UNIQUE VISITS. ABOUT A HUNDRED
THOUSAND A YEAR YEAR. A VISIT TWICE
AS MANY PER VISITOR MEANS
VISITOR IS DOING TWO THINGS.
THAT IS ONE WAY TO THINK ABOUT
IT. ON AVERAGE. ANOTHER METRIC
YOU CAN LOOK AT HOW MANY 3-D
STRUCTURE FILES ARE DOWNLOADED
EVERY YEAR. SO THEY HAVE GONE TO
OUR SITE NOT JUST TO LOOK ATE AT
IT, YOU CAN GO TO SITE AND LOOK
AND MODEL AND SAY THAT IS COOL
BUT YOU CAN DOWNLOAD PDB FILE
AND PLAY WITH IT WITH CHIMERA OR
WHATEVER YOU LIKE. NOW WE ARE UP
TO JUST OVER ABOUT 14,000 A
YEAR. THAT'S A SIGNIFICANT
INCREASE AND VERY SATISFYING
NUMBER TO SEE PEOPLE ACTUALLY
USE, THESE ARE NOT STATISTICS
THAT INCLUDE RECENT API STUFF OR
URL THIS IS PEOPLE GOING
QUAINTLY AND CLICKING AT THE EBB
WEBSITE. IN TERMS OF BREADTH OF
USE, THIS IS A SUMMARY OF THE
PAST FIVE YEARS. LOOKS GREAT.
BUT IN FACT DOESN'T LOOK MUCH
DIFFERENT YEAR-TO-YEAR. ROUGHLY
HALFWAY SPLIT BETWEEN NORTH
AMERICA AND EUROPE WITH QUITE A
BIT OF SITES IN ASIA. IT IS
INTERESTING IF I HAD MORE TIME I
WOULD SHOW THIS YEAR-TO-YEAR AND
YOU CAN SEE HOW IT SHIFTS AS
DIFFERENT GLYCOSCIENTISTS
DEVELOP INTEREST IN DIFFERENT
AREASAREAS. SOUTH AMERICA IS DIVERSE
AN SCATTERED. IT IS FUN. BUT IT
SHOWS ONE OF THE VERY EXCITING
THINGS ABOUT AUTONOMOUS WEB
TOOLS IS THEY ARE GLOBAL, NO
DOUBT. THIS API I MENTIONED THIS
TOOK AN -- AND IS ONGOING, TOOK
HUGE AMOUNT OF EFFORT BECAUSE
THE WEBSITE WASN'T DEVELOPED
WITH THIS IN MIND AT FIRST
WHATSOEVER. THE WEBSITE IS OLD.
AND THE FROM INFRASTRUCTURE IN
IF WEB DEVELOPMENT AND CODING,
CHANGES ALL THE TIME. AND
ANYBODY WHO IS DOING WEB
DEVELOPMENT OR HOSTING WEB TOOLS
FACES CHALLENGES THIS WAY
BECAUSE EVEN IF YOU SAY MAKE ONE
WAY AND KEEP IT STATIC YOU
CAN'T. BECAUSE THINGS LIKE
SECURITY PATCHES REQUIRED AND
THAT MAY REQUIRE UPDATE OF THIS,
OR THAT. SO YOU GOT TO
CONSTANTLY KEEP ON TOP OF STUFF.
WHAT WE DECIDE TO DO IS CREATE
PROPRIETARY SOFTWARE CALLED GEMS
AND GLYCAN MOLECULAR LIBRARY,
THAT KEEPS SCIENCE SEPARATE FROM
WEBSITE SO WE CAN HIRE C PLUS
PLUS PROGRAMMERS OR PYTHON
PROGRAMMERS AND THEY DON'T HAVE
TO LEARN CARBOHYDRATE CHEMISTRY,
THEY CAN BE C PLUS PLUS
PROGRAMMERS. LOVE TO RECRUIT
MORE. ALL THESE I GET SHUTTLED
TO VARIOUS COMPUTERS, IN MY MIND
IT GOES TO PARTICULAR COMPUTER
AND NO, NO IT GOES TO SWARM AND
WE FARM IT OUT. OKAY NOT MY
WHEEL HOUSE. WE HAVE TWO GUI'S
NOW, THIS IS AN ODD THING THIS
IS THE OLDER WITHIN WE CALL IT
BUILDING LEFT TO RIGHT. BECAUSE
YOU START ON THE GGALPNAC OR THE
NON-REDUCING TERMINUS OF THE
GRABBING TOE PYRANOSE BETA 14
LINKED. GAL BETA FORWARD GLCNAC
THAT IS HOW YOU POINT CLICK AND
BUILD IT. THE CHALLENGE IS WHEN
YOU HAVE TO BIOTYNARY WHAT
SHOULD I START IT DOESN'T MATTER
BUT THEY DON'T KNOW THAT SO THE
PROGRAMMERS SAID TO ME WOULDN'T
IT MAKE MORE SENSE TO START
REDUCING TERMINUS BECAUSE THERE
IS ONLY ONE AND BUILD THE OTHER
WAY? IT MIGHT MAKE MORE SENSE SO
THAT IS WHAT WE DID. WE HAVE A
DIFFERENT BUILDER HERE YOU START
SAYING OKAY WE HAVE MAN ATTACHED
TO GAL AND THEN YOU SPECIFY
LINKAGE THEN ANOTHER SPECIFY
THEN RIGHT TO LEFT. THIS BREAKS
MY HEAD BECAUSE I'M AN OLD
SCHOOL CARBOHYDRATE CHEMIST LEFT
TO RIGHT. BE THIS IS HOW SAY B
BASED CHEMISTRY IS DONE. YOU GO
THE OTHER WAY. THERE ARE
ADVANTAGES. I WOULD LIKE
EVERYBODY HERE TO TAKE THE TIME
TO GO O TO OUR SITE AND TRY LEFT
TO RIGHT AND RIGHT TO LEFT
BUILDER AND SEND ME AN EMAIL.
SAY I HATE THIS, I LIKE THAT. WE
ARE NOT SUPPORTING BOTH. ONE IS
GOING TO GO THE WAY OF THE DODO.
I HOPE IT IS NOT MINE I WILL LET
COMMUNITY DECIDE ON THAT. OTHER
THINGS WE DO, I'M PERSONALLY
EXCITED ABOUT IS BUILDING MODELS
OF GLYCOPROTEINS. WE DO THIS ALL
THE TIME BUT IT IS CHALLENGING
TO DO. I WILL SHOW A COUPLE OF
ISSUES. FIRST SHOW YOU WHAT WE
WANT TO HAVE. USER SHOULD BE
ABLE TO UPLOAD PBB STRUCTURE, IT
ALSO CAN BE THEORETICAL
STRUCTURE, ALPHA FOLD, A TOTALLY
HYPOTHETICAL HOMOLOGY MODEL. I
KNOW THERE'S GLYCOSYLATION OF
SITES OR GO TO PROGRAM THAT SAYS
PREDICTED GLYCOSYLATION. PUT
THESE GLYCANS ON AT THESE SITES
SO WE DO THAT. AND IT THEN
SCREEN RATES STATIC IMAGE OF C
ON THE BOTTOM BUT AND GIVES BANG
AND THAT IS -- WE CAN DO THAT
NOW. WHAT IS NICE TO DO IS HAVE
THIS FED INTO AUTOMATED TOOL FOR
MD EMULATION SO YOU GET A SENSE
OF HOW MUCH MOTION IS GOING ON
HERE. THAT IS A COSTLY THING. WE
WILL SEE HOW THAT GOES. THIS IS
NOT AS PULLY IMPLEMENTED AT THE
API STAGE AS WE LIKE IT YET BUT
IT IS GETTING THERE. SO YOU
WOULD PASS TO THE TOOL HERE IS
THE PROTEIN HERE IS
GLYCOSYLATION SITES, HERE IS
GLYCANS I WANT AT EACH SITE. ONE
THING THAT COMES FROM THIS, IS
WHEN YOU TALK THE PEOPLE WHO DO
GLYCOPROTEOMICS WOW IT FORCES
THEM TO TELL ME WHAT GLYCAN THEY
WANT AT EACH SITE. THAT FIRMLY
REINFORCES THE CONCEPTS
GLYCOPROTEINS ARE MIXTURES OF
GLYCOFORMS. YOU CAN'T HAVE
MIXTURE OF GLYCANSES ON SITE AND
MODEL IT. YOU PICK ONE AND MODEL
IT. THERE ARE SOME CHALLENGES
HERE. WHEN WE INITIALLY BUILD A
STRUCTURE COMPUTER DOESN'T KNOW
HOW TO PUT THE GLYCAN. IT'S JUST
A PROTEIN AND WE SAY STICK
GLYCAN HERE. SO WE ARBITRARILY
ORIENT GLYCANS ON SURFACE AND IF
YOU LOOK CLOSELY AT THIS YOU
WILL SEE THAT SOME OF THE GLYCAN
STRUCTURE ARE PENETRATING
PROTEIN SURFACE. OR BANGING INTO
EACH OTHER. THESE ARE WHAT WE
CALL ARTIFICIAL COLLISIONS OR
OVERLAPS, OBVIOUSLY THEY HAVE TO
BE RESOLVED IT IS NOT A -- IT IS
NOT A REAL THING. SO WE GO
THROUGH SOME SORT OF ALGORITHM
WHERE WE TWIST THESE AROUND
AMONG ALLOWABLE CONFIRMATIONS OF
THE ASPARAGINE SIDE CHAIN OR
ALLOWABLE INTERNAL ANGLES UNTIL
WE FIND SOLUTION THAT IS
CONSISTENT WITH EXPERIMENTAL AND
ENERGY COAGULATION THEN WE CAN
START OUR SIMULATION. THIS IS
REALLY EASY ON SMALL THING LIKE
THIS BUT IF YOU GET TO SAY GP
120 WITH HUGE DENSITY OF GLYCANS
IT BECOMES A VERY INTERESTING
ALGORITHMIC CHALLENGE TO FIGURE
THE BEST WAY TO RESOLVE THESE
OVERLAPS. THIS IS A FUN
COMPUTATIONAL PROJECT. IN TERMS
OF MODELING, I LIKE TO SAY WE
HAD A SPIKE DURING COVID-19
BECAUSE EVERYBODY AND THEIR
COLLEAGUE WAS MODELING SPIKE
PROTEIN. A LOT WERE USING OUR
TOOL YOU CAN SEE BY MONTH IN
APRIL OF 2020 ALMOST A THOUSAND
DOWNLOADS OF MODELS OF SPIKE
WITH GLYCANS ON IT. FEW OF THOSE
WERE FROM OUR GROUP AS YOU CAN
SEE ON THE LEFT MOSS PLOT OF
STRUCTURE OF THE SPIKE PROTEIN
WHERE WE HAVE COLORED THE
PROTEIN IN RED ACCORDING TO
ANTIBODY ACCESSIBILITY AND THEN
THE GLYCANS AS THE MOSS
STRUCTURES. WE HAVE A LOT OF
COLLABORATORS. THIS HAS BEEN
WIDELY EXTERNALLY EVALUATED
CROSS VALIDATED, I ENCOURAGE
PEOPLE TO SEND PEOPLE INTO MY
GROUP TO GET TRAINING RATHER
THAN JUST A COLLABORATOR. I LIKE
THE IDEA OF TEACHING SOMEONE TO
FISH RATHER THAN GIVING THEM A
FISH. AND I THINK IT PROP GATES
TECHNOLOGY BETTER THAT WAY. THAT
IS IT FOR US. THIS IS A BIG
TEAM PROJECT. WE HAVE WEB DEV
AND PYTHON DEVELOPERS C PLUS
PLUS PROGRAMMERS, ALL OF THIS
SUPPORTED MOST GRATEFULLY FROM
THE NIH. SO I THANK YOU GUYS FOR
YOUR PATIENCE AND LOOK FORWARD
TO SEEING YOU AT DINNER.
[APPLAUSE]
>> THANK YOU. AN ANNOUNCEMENT.
>> I JUST HAVE ONE BRIEF
ANNOUNCEMENT SO ONE OF THE
THINGS THAT'S BEEN SO COOL ABOUT
THIS MEETING IS JUST SEEING ALL
THE BEAUTIFUL SCIENCE AND WHERE
THINGS ARE GOING AND IT IS NICE
WE HAVE ALWAYS BEEN TALKING
ABOUT OUTSIDE RECOGNITION SO I
WANT TO JUST MENTION SOME
OUTSIDE RECOGNITION FOR SOMEONE
IN OUR AUDIENCE TODAY. WE JUST
FOUND OUT OR SHE JUST FOUND OUT
AND IT IS ALSO ONLINE THAT LINDA
SHAY WILSON HAS BEEN ELECTED TO
THE NATIONAL ACADEMY OF
SCIENCES. SO I THINK --
[APPLAUSE]
WE SHOULD ALL CONGRATULATE LINDA
AND MAYBE SOME OF YOU WHO ARE
READY FOR DINNER WE CAN TOAST TO
HER SUCCESS. CONGRATS LINDA