WELCOME TO THE PROTIG LECTURE SO WHAT WE'RE GOING TO TALK ABOUT TODAY IS SOME WORK THAT WAS DONE IN MY LAB. WHILE THE WORK WAS DONE BY A POST DOC IN THE LAB, WHO IS NOW WORKING AT FDA -- WHO RUNS THE INSTRUMENT IS BILL DENNIS. AS IN NIANY FEDERAL EMPLOYEE, I HAVE TO MAKE A DISCLAIMER, THAT THIS WORK IS MY OPINION ONLY AND NOT THE OFFICIAL POSITION OF THE ARMY. AND IF I DO MENTION ANY COMMERCIAL NAMES, IT'S NOT A CITATION OF THAT MATERIAL. THIS WORK WAS FUNDED BY THE ARMY ARMY, THE MILITARY OPERATIONAL MEDICINE RESEARCH PROGRAM OF THE U.S. ARMY. SO I AM GOING TO START OFF TODAY TALKING A LITTLE BIT ABOUT THE PROGRAM THAT DEPUTY DIRECTOR OF THE U.S. ARMY FOR ENVIRONMENTAL HEALTH RESEARCH. AND THEN I'LL TALK ABOUT HOW ON THE PROTEINS IN THE CELL LINES AND SOME FUTURE DIRECTIONS. SO, I AM THE DEPUTY DRAWER OF THE ENVIRONMENTAL HEALTH PROGRAM AT THE U.S. ARMY CENTER FOR ENVIRONMENTAL HEALTH RESEARCH. WE HAVE 4 AREA AREAS, OR GROUP CENTERS. IT'S A SMALL PROGRAM, BUT WE KIND OF HAVE MULTIPLE FOCUSES. WE HAVE SOME ADMINISTRATIVE THAFKZ THAT WE CAN DO AND THE NORMAL DOCUMENTATION ITSELF AND GRANTS AND AGREEMENTS. PROBABLY THE LARGEST FOCUS AREA ON RESEARCH IS LOOKING AT ADD ADVERSE HEALTH EFFECTS. AND ONE OF OUR BIG FOCUSES HERE IS TRYING TO IDENTIFY BIOMARKERS THAT CAN DETERMINE WHETHER SOLDIERS WHO HAVE BEEN EXPOSED TO CHEMICALS ARE HAVING ADVERSE HEALTH EFFECTS AS A RESULT OF THOSE EXPOSURES. WE ARE -- THIS POINTER'S GOING TO BE A PROBLEM. THE NEW PRAYAREA WE'RE STARTING TO GET INTO IS NAN ORESEARCH AND IT'S A NEW BRANCH WE'RE WORKING WITH AT GANT. THE HOST PROVINCE RESPONSE IS THE MAIN AREA AND WE'RE WORKING WITH HEAT STRESS AND WE'RE TRYING TO BUILD A LARGER AREA OF HEAT STRESS. ANOTHER BRANCH WHY NOT WITHIN THE ENVIRONMENTAL PROGRAM IS DOING EXPOSURE ASSESSMENT, WHICH IS PRIMARILY RELATED TO WORK THAT HAD BEEN GOING ON, SOME LEGISLATIACY STUFF. BUT THESE ARE WHERE WE CAN TEST WHETHER OR NOT MATERIAL ARE PRESENT. SO WE'VE HAD A ROBUST TOXICOLOGY LAB AND WE'VE DEVELOPED SOME SENSORS THAT AWE'LL BE ABLE TO TEST DRINK IING WATER THAT MIGHT HAVE CHEMICALS IN IT. IN FACT, THE DC WATER SUPPLY IS PROTECTED BY ONE OF THE ONES THAT WAS CREATED. WE HAVE A NEW STARTUP PROJECT, WHERE WE'RE LOOKING AT HOW MI MICRMICRO BIORESPONSE EXPOSURES, WHICH IS A NEW, INTERESTING AREA. WE KNOW THAT THE MICRO BIOME HAS A MAJOR EFFECT ON HEALTH, AND WE ALSO KNOW THAT THE MICRO BIOME CAN BE AFFECTED BY TOXIC EXPOSURES. SO WE WANT TO LOOK AT SOME TOXIC EXPOSURES TO LOOK AT HOW THEY CHANGE AND MEASURE HOW THAT CHANGE MIGHT BE A GOOD INDICATOR OF WHAT PEOPLE HAVE BEEN EXPOSED TO. AND THEN WE ALSO HAVE, WITHIN OUR GROUP, SOME CORE CAPABILITIES, KIND OF THE INFRA INFRASTRUCTURE THAT LETS US DO OUR WORK. SO FOR A SMALL GROUP, WE ACTUALLY HAVE A LOT OF HIGH-END EQUIPMENT. THE ARMY DOES A PRETTY GOOD JOB OF FINDING MONEY AT THE END OF THE YEAR SO WE CAN GET SOME IN INFRASTRUCTURE? IN PLACE. SO WE HAVE TRANSCRIPT ELEMENTS, FOR EXAMPLE, PROTEOMICS CAPABILITIES AND KCHEMISTRY SUITE SUITE. YOU HAVE TO MEASURE THE CHEMICAL USING THE WATER SYSTEMS, SO WE CAN MEASURE MOST CHEMICALS WE USE IN-HOUSE. AND THE AQUATICS TRADITIONAL, WE JUST HAD A BO VAR IRIAN XREET EED AND WE DON'T HAVE ANIMALS. WE SHOULD HAVE ANIMALS WITHIN THE NEXT SEVERAL MONTHS. LIKE I SAID, IT'S NOT A BIG PROGRAM. ALL TOTAL, WE HAVE ABOUT 13 PERSONNEL. HALF OF THEM ARE PH. D LEVELS, POST DOCS, OR HIGHER. WE ALSO HAVE ONE PH. D WHO IS IN OUR SMART PROGRAM, WHO IS FINISH ING HIS P SHELTHD IN MAY AND WILL BE STARTING WITH US. AS I SAID, WITH OUR EQUIPMENT, WE ARE PRETTY WELL STOCKED FOR THAT. WE HAVE AINFERMEORMATATTICS SYSTEM AND ONE OF THEM ALLOW S US TO DO MICROW ARRAY S. FOR PROTEOMICS, WE HAVE VELO S, WHICH THE WORK WAS DONE ON OUR INSTRUMENT. WE HAVE A WATER 2, WHICH DOES QUANTITATIVE WORK ON THAT. AND WE HAVE A ZEBBO P Q, IT'S A TYPE OF SYSTEM WHERE, INSTEAD OF HIRYDROFOIL S THEY HAVE STACKS THAT ALLOW YOU TO MANIPULATE THEM A LITTLE BETTER. FOR KCHEMISTRY, WE HAVE -- WHEN WE THINK OF CHEMICAL, WE'RE DOING METALS, GCS, SO WE CAN QUANTITATIVE MOST OF THE CHEMICALS WE WORK WITH. AND TO SUPPORT THAT, WE ALSO HAVE MOSTLY THE SOFTWARE IS COMMERCIAL SOFTWARE, WHILE OPEN SOURCE IS AVAILABLE FOR THINGS WE DO WHEN WE NEED TO, THE INFRA INFRASDPRURT REQUIREMENTS GETTING OPEN-SOURCE RUNNING IS MORE EXPENSIVE THAN BUYING COMPLESHL SO WE MOSTLY USE COMMERCIAL SOFTWARE. AND AS I SAID, THESE ARE THE FISH AND THE RODENTS WE'LL BE ABLE TO USE. WE ALSO HAVE A SEQUENCE R USING R RNA C AND WORK WITH THAT. WE HAVE THESE RESOURCES. WE HAVE STAFF TO RUN THEM. WE ALWAYS LOOK FOR COLLABORATES S. SO PEOPLE -- COLLABORATIONS. SO PEOPLE WHO HAVE PROJECTS THAT MIGHT BE ABLE TO USE SOME OF THE EQUIPMENT, WE'RE ALWAYS LOOKING FOR PEOPLE WHO MIGHT BE INTERESTED IN DOING COLLABORATES S WITH US. -- COLLABORATIONS WITH US. SO THAT IS THE INFRASTRUCTURE SIDE, AND THIS IS MORE OF THE LAYOUT OF THE RESEARCH. SO THE WAY OUR RESEARCH IS FUND FUNDED IS HIKERARCHY IS ACTUALLY CALLED "TASK AREA."^ SO THE TASK AREA IS KIND OF THE OVERARCHING IDEA OF WHAT'S RESEARCH -- WHAT RESEARCH WE'RE LOOKING TO DO. AND WHAT WE'RE TRYING TO DO IS USING EXPERIMENTAL COMP PUTE ATION AL APPROACHES TO CHARACTER IZE ADVERSE EFFECTS RESPONSIBILITIRESPONSES. SO WE'RE NOT MEASURING FOR CHEMICALS. A LOT OF TIMES WE'RE LOOKING AT HOW THE ORGANISM RESPONDS AND HOW HUMANS RESPOND. WE DO HAVE RODENTS BUT HOW THE ORGANISM IS GOING TO RESPOND OR HOW HUMANS ARE LIKELY TO RESPOND RESPOND. SO WE'RE LOOKING AT MARKERS THAT ARE INDICATING THEIR HEALTH EFFECTS BY THEIR OWN PROTEINS AND DNA MOLECULES. SO WITHIN THAT TASK AREA, WE HAVE 3 MAIN GROUPS OF RESEARCH. THE LARGEST IS THE BIOMARKERS OF APOP TASSY AND HEALTH EFFECTS. WE HAVE MULTIPLE COLLABORATIONS SET UP FOR THIS, SO DHSI IS A HIGH-PERFORMING GROUP THAT THE ARMY HAS AND WE HAVE A COLLABORATION WITH THE NAVY RESEARCH LAB FOR THIS. AND THIS RIGHT NOW, WE'RE FOCUSED ING ON SOME BIOMARKERS WITH LIVER FIBROSIS. BUT THAT CAN BE SPREAD OUT. WE MIGHT DO KIDNEY AS WELL. BUT ONE OF THE FOCUSES OF THAT PROJECT IS STARTING USING PUBLIC PUBLICLY DATASET AND A DATABASE, WHICH IS A LARGE MICROW ARRAY RE REPOSITIORY, WHERE THERE WAS TOX TOXIC EXPOSURES DONE, PA PATHOLOGIES DONE. AND THEY CAN LOOK BACK AT THOSE DATA AND TRY TO MINE THEM AND GET BETTER OZ IDEAS OF WHAT ARE GOOD TARGETS TO BE LOOKING AT WHEN WE'RE DOING OUR OWN EXPERIMENTS. WE HAVE TOOLS TO WORK WITH, BUT WE'RE ALSO TRYING TO HAVE AN IDEA OF WHAT WE'RE LOOKING AT AHEAD OF TIME TO TRY TO HELP FOCUS OUR EFFORTS. WE HAVE A MID-SIZED PROJECT ON HEAVY METAL TOXIC IITY, AND THIS IS A PROJECT THAT HAS BEEN GOING ON FOR SEVERAL YEARS AND THIS IS TRYING TO BE A CROSS SPECIES EX EXTRAPOLATION. WE'VE DONE METAL EXPOSURES IN FISH AND LIVER RODENT CELL LINES AND HUMAN CELL LINES. AND THE IDEA IS ULTIMATE LY TO TRY TO DO SOME SORT OF COMP PUTE ATIONAL EXTRAPOLATION TO GET A BETTER IDEA OF WHAT IS HAPPENING IN HUMANS. AND THE NEWEST PROJECT IS THE MI MICRO BIOME PROJECT AND MOSTLY THE WORK START ED THED THIS FISCAL YEAR. WE'RE WORKING PRIMARILY WITH SAM REDD, U.S. ARMY RESEARCH INSTITUTE FOR INFECTIOUS DISEASE DISEASE. THEY DO A LOT OF BACTERIAIAL WORK SCAAND WAYS OF CHARACTERIZING GENOME. SO WE'RE USING SOME OF THEIR INSTRUMENTATION AND COLLABORATE COLLABORATING WITH THEM TO BE ABLE TO CHARACTERIZE THE MICRO BIOME, ONCE AGAIN, WORKING WORKING WITH D SHELTHSI, TO DO SOME OF THE COMPUTATION AAL WORK. SO THAT'S THE PROGRAM THAT I AM THE DEPUTY DIRECTOR OF. WE'RE ALWAYS LOOKING FOR COLLABORATIONS. WE HAVE INSTRUMENTS AND SOME OF THEM ARE RUN FULL-TIME AND SOME OF THEM AREN'T. SO I'M ALWAYS LOOKING FOR OPTION OPTIONS. SO IF PEOPLE HAVE IDEAS AND HOPE HOPEFULLY SOMEWHERE IN OUR TRACT OF RESEARCH, WE'D BE HAPPY TO TALK WITH YOU. ALL RIGHT, SO MOVING ON TO THE A ADDUCTS. SO DIPROVE VOST IS AN OR KNICK PESTICIDE -- ORGANIC PESTICIDE. I'M TRYING TO BRING THIS UP HIGHER. ALL RIGHT, THAT'S BETTER. ALL RIGHT, SO DIPROVE AATE IS AN OR G ORGAN IC PESTICIDE CHARACTERIZED BY THE FOPHOSPHOFOSPHOSPHATE GROUP, AND PRIMARILY THE TARGET IS TO INHIBIT AN ENZYLE THAT PROCESSES THE NEWUROTRANSMITTER. THIS PESTICIDE IS ABLE TO BIND THE ACTIVE SITES AND BLOCK THE SEER IING SO IT CAN INHIBIT ASEAE THEIL COLONASTRASS IS. WHEN YOU BLOCK ASEE THEIL COLON ASTRAITS, YOU CAN'T BREATHE, YOU DIE. THERE ARE OVER 200 -- 250,000 DEATHS A YEAR. THERE IS MANY LOWER-LEVEL EFFECTS AND OTHER SYMPTOMS. WE HAVE LOW-LEVEL CHRONIC EXPOSURES, AND NOT ALL THEMES SYMPTOMS -- ALL THESE SYMPTOMS CAN BE ATTRIBUTED TO THIS. AND THERE HAVE BEEN SOME OTHER TARGETS IDENTIFIED. BUT IT'S BEEN A SLOW SPRAYS -- PROCESS. AND THIS IS A LITTLE BIT MORE COMPREHENSIVE THAN SOME OF THE OTHER ONES THAT HAVE BEEN PUBLISHED. BUT SO FAR, WHAT ARE THE CHRONIC EFFECTS THAT ARE CAUSED WITH WHAT PROTEINS ARE INTERACTING WITH? IT TURNS OUT, SINCE THIS IS A REACT IIVE CHEMICAL, IT CAN REACT WITH SER IINES AND TIRYROSINES ON A VARIETY OF PROTEINS. AND THE QUESTION IS WHAT PRO PROTEINS IS IT INTERACTING WITH AND WE HAVE NEW TARGETS THAT MIGHT BE THE CAUSES OF ADDITIONAL ADVERSE EFFECTS FROM DICOLORIX EXPOSURE. SO WE WENT LOOKING FOR THESE A ADDUCTS. SO THIS IS THE DICLOFB IS MOLL MOLECULE. WHEN IT FORMS AN ADDUCT, IT LOSE LOSES THE COLHLORINE SIDE OF THIS. SO THIS IS WHERE IT ATTACHES TO THE PROTEIN. SO THE ORIGINAL NORFORM OF IT HAS 2 METAL GROUPS ON IT. THEY GO THROUGH A PROCESS CALLED "AGING."^ SO AS IT AGES, IT LOSE LOSES ONE OF THE METAL GROUPS. -- METHYL GROUP. ONCE IT LOSES THAT METHYL GROUP, IT BECOMES MORE RESIST AANT TO TREATMENT. SO NORMALLY WHEN YOU ARE EXPOSED TO AN OP AGENT, UB BE TREATED WITH ENZYMES TO REACTRESS -- RE REACTIVATE AND ONCE IT GOES THROUGH THIS AGING PROCESS, IT'S MORE SUSCEPTIBLE TO REVERSAL. WE LOOK FOR PEPTIDES THAT HAVE BEEN TREATED WITH DICOLOR OOSE. SO KIND OF A SPLIT STRATEGY HERE. FIRST THING WE WANT TO DO IS TRY TO IDENTIFY TARGETS. WE USE 2 DIFFERENT APPROACHES FOR IDENTIFICATION. ONE WAS JUST EXPOSING CELL LICE LYSASE TO THE DICLOFB OOSE AND THE ANALYSIS TO TRY TO FIND THE LOCATIONS WHERE THE ADDUCTS WERE FORMED. AND THE OTHER WE USED WAS USING IT IN AN ASSAY WHERE THERE IS AN AS PCHP, WHICH CAN ALSO BINDS TO THE SAME SITES AS OBB AGENTS. SO IT'S KIND OF A COM PEW TATATION ASSAY. AND WE DID CONFIRMATION. SOME OF THE PROTEINS WE IDENTIFIED IN THESE ASSAYS ARE PURIFIED PROTEINS AND EXPOSED THEM DIRECTLY TO VERIFY THE A ADDUCTS. BUT WE DID ASSAYS TO SHOW THAT THE DICLOFB OOSE NOT ONLY BOUND ED TO FORM THE ADDUCTS BUT INHIBIT ED THE DICLIVAT TIC ACTIVITY. WE DID THAT WITH PROTEINS AND CELLS THAT WERE TREATED WITH THE DICLOFB OOSE. SO BIOTICIN IS BASICALLY A CHEMICAL THAT HAS A GROUP THAT IS VERY SIMILAR TO AN OP GROUP. IT CAN FORM ADDUCTS ON SITES THAT ARE SIMILAR TO THE SAME SITES. AND ON THE OTHER END VITEOF THE MOLL MOLECULE IS A BIOTTIN. SO WE USE IT FOR PROTEINS THAT HAVE BEEN MODIFIED WITH THE FD BIOTIN. IF WE DON'T TREAT IT AND WE EXPOSE PROTEINS TO THE F BIOTIN AND PULL THEM DOWN, ALL THE PRO PROTEINS IT INTERACTS WITH WILL BE PULLED DOWN. AND THEN WE CAN ANALYZE THE QUANTITY OF THOSE PROTEINS AND KNOW WHAT ARE THE TARGETS OF FP BIOTIN. IF ANY OF THE TARGETS OVERLAP, THOSE TARGETS, WE CAN TRY TO IDENTIFY THEM BY LOOKING FOR -- BY BLOCKING THE FT BIOTIN REACTION BY TREATING THE PRO PROTEINS WITH DIPROVE OST. ONCE IT'S FORMED, IT'S BLOCKED WITH FT BIOTIN INTERACTING WITH THAT SITE. SINCE IT'S BLOCKED, YOU PULL DOWN THE ONES THAT HAVE BIOTIN A ATTACHED TO THEM. SO ANY OF THEM THAT HAVE BEEN BLOCKED ARE GOING TO BE MISSING IN THESE SAMPLES. SO IF WE DO A QUANTITATIVE COMPARISON THEN OF THE PROTEINS WE PULL DOWN BEFORE WE TREATED, THE ONES THAT ARE IN LOWER ABUNDANCE IN THE TREATED SAMPLES ARE THE ONES THAT ARE TARGETS FOR FT BIOTIN AND POTENTIAL TARGETS FOR BICHLOROIS. FOR QUANTIFICATION, WITHIN THE LAB WE USEW Ö APPROACHES FOR QUANTIFICATION. BUT THIS EXPERIMENT WAS DONE WITH LABEL-3 QUANTIFICATION, AND WE HAVE A SOFTWARE PACKAGE CALLED ADVANTAGE LC MCHM S, WHICH IS A PACKAGE THAT WAS ORIGINALLY THE EARLIER VERSION OF GELS. YOU RUN YOUR MASS SPECK AND TUZZ -- IT DOES AN ALIGNMENT AND A ALIGNS ALL YOUR PEAKS. AND ONCE THEY'RE ALIGNED, YOU GET QUANTIFICATION. IT WILL GIVE YOU A BASELINE SCORE, SO YOU DON'T HAVE MUCH MISSING DATA, JUST BECAUSE YOU DIDN'T GET AN MSMM ON THAT RUN OR SOMETHING. SO WE USE THAT FOR QUANTITY QUANTITIFICATION AND THESE SAMPLES WERE RUN ON THE VELOS. SO THESE WERE CELLS THAT WERE UN UNTREATED. WE JUST EXTRACTED THE PROTEINS. AND THEN IN A TEST TUBE, WE CREATED -- TREATED THEM WITH DI DICHLOROIS OOSE. WE USED 600 MICRO MORAL FOR 2 HOURS. RAN IT ON THE VELOS. AND THEN WE IDENTIFIED PROTEINS THAT ARE CHANGING THAT HAD TO RELEASE 1.5 PULL-DOWN. SO THESE ARE STATISTICALLY RELEVANT. SO ANY PROTEIN THAT WAS DOWN DOWNREGULATED, THESE ARE THE ONES THAT HAVE THE FP BIOTIN BLOCKED. YES? >> I THINK THAT WOULD BE TRICKY TRICKY, RIGHT? >> INAUDIB[INAUDIBLE] >> THAT IS A LITTLE BIT OF A COLLAGE. THERE ARE NORMALIZATIONS WITHIN THE SOFTWARE BUT THERE IS A BIAS BECAUSE NORMALLY WHEN YOU ARE DOING QUANTITIFICATION, SOME ARE IN ABUNDANCE. BUT HERE, NOTHING SHOULD BE GOING UNIN ABUNDANCE. EVERYTHING SHOULD BE GOING DOWN, SO THERE IS POTENTIALLY A BIAS TO THE DATA. -- DATA. >> SO THERE ARE SOME ISSUES. SO THAT'S A CONCERN THAT WE HAVE HAVE. OTHERWISE THERE IS ANOTHER CONCERN THAT WE HAVE THAT DI DICHLOROIS CAN CAUSE PESIP TATE, TOO,, SO WE HAVE TO BE CAREFUL WITH THAT. I'VE NEVER WORKED OUT EXACTLY WHY. PROBABLY IT'S A P SHELTH ISSUE, BUT WE'RE HAVING ISSUES AT THE HIGH DICHLOROIS CONCENTRATIONS. EVERYTHING GOES UP IN ABUNDANCE. WE ENDED UP IDENTIFYING 511 PRO PROTEINS IN THE ANALYSIS, AND 70 OF THESE PROTEINS HAD A LOWER ABUNDANCE. REALLY HIGH. >> INAUDIB[INAUDIBLE] >> THEY ARE PROBABLY NOT VERY BIOLOGICAL LY RELEVANT. THE 5 MORALS ARE HIGH CONS CONCENTRATION. BUT WHAT WE'RE DOING HERE IS WE'RE TRYING TO FIND TARGETS. SO WE'RE STARTING AND GETTING FUTURE DIRECTIONS TO BE ABLE TO DO MORE SENSITIVE ASSAYS. WE'RE REALLY JUST DRIVING REACTION. >> EVEN THE NON-COVALENT -- >> NO, THESE ARE COVALENT A ADDUCTS. >> INAUDIB[INAUDIBLE] ARE YOU JUST LOOKING FOR OTHER -- >> SO IT'S LOOKING FOR COVALENT LY MODIFIED PEPTIDES. SO IN THIS ASSAY WE'RE NOT LOOKING FOR COVALENT LY MODIFIED PEPTIDES. WE WOULDN'T SEE IT IN THE SAMPLES WE'RE LOOKING AT. HERE, IT'S QUANTIFICATION-ONLY. WE'RE LOOKING FOR PROTEINS THAT ARE LOWER IN ABUNDANCE WITH THE MOST LIKELY EXPLANATION IS THAT IT'S BLOCKING THE FT BIOTIN BOND BONDING SITES. THOUGH IT'S ALSO -- THERE IS A LOT OF OTHER THINGS THAT COULD HAPPEN TO LOWER THE ABUNDANCE, BUT THIS IS A TARGET LIST. IT'S NOT -- AND AS I'LL SHOW YOU YOU, ONE OF THE ONES IN THIS LIST DOESN'T GO THROUGH, WE NEVER FOUND ADD DUCTS. THIS LIST IS ENRICHED FOR DI DICHLOROIS TARGETS. AS I SAID, THE FALSE POSITIVES AND BINDING PROTEIN COMPLEXES ARE QUANTITIFICATION. AS I'LL SHOW YOU LATER, WE TOOK 2 OF THE PROTEINS AND GOT LDH WHICH WERE IDENTIFIED IN THIS LIST, AND WE WERE UNABLE TO FIND ADDUCTS USING PURIFIED PROTEIN A ADDUCTS ON THEM. WE MIGHT HAVE NOT SEEN THEM, BUT THIS LIST IS EN -- IN RICH DI DICHLOROIS. THE OTHER APPROACH WAS THE DIRECT APPROACH, WHERE WE JUST CREATED THESE PROTEINS FROM A CELL EXTRACT USING THE SAME CON CONCENTRATIONS. AND WE JUST RAN THEM AND LOOKED FOR MODIFICATIONS ON SER ININE, THREONINE OR TIRYROSIGN, WHICH THAT ARE THE APPROPRIATE MATCH. SO IN THIS CASE, WE'RE LOOKING FOR THEM TO SOUTHERNED -- CORRESPOND WITH THE ADDUCT. IN THIS ANALYSIS, THERE IS JUST OVER 2,000 PEPTIDES THAT WERE IDENTIFIED WITH HIGH-CONFIDENT SCORES AND 53 OF THESE PEPTIDES HAD ADDUCTS. AND THERE WAS DIFFERENT ADDUCT COMBINATIONS SO I'LL GET INTO WHAT THAT MEANS. SOMETIMES IT MIGHT HAVE BEEN BOTH THE AGED AND UNAGED VERSION OF THE ADDUCTS. SOME OF THE PEPTIDES ACTUALLY HAD MORE THAN ONE SITE ON THEM. SO, ONE OF THE THINGS THAT TURNED UP IS THAT WE HAD MULTI MULTIPLE PEPTIDES THAT HAD MORE THAN ONE ADDUCT SITES IDENTIFIED IDENTIFIED. IN SEVERAL CAYS IN SEVERAL CASE CASES, THIS IS ONE OF THE EXAMPLES WHERE THERE WAS TIYRO TYROSIGN AND THREONINE ADJACENT TO EACH OTHER. AND THEY MIGHT HAVE CLAIMED THAT THE ADDUCT WAS ON THE TIYROSIGN OR IT MIGHT HAVE BEEN ON THE THREONINE. SO THE QUESTION IS CAN WE TRULY DIFFERENTIATE WHICH WAS WHICH. FOR ANYTHING THAT HAD 2 ADDUCTS ON IT, I WENT THROUGH THE LIST AND MADE SURE THAT THERE WAS A GOOD PRESENT THAT THIS WAS BETWEEN THE 2 SITES. IT HAD TO HAVE AN ASSAY OF LESS THAN 200 PARTS PER MILLION AND PROBABLY AROUND 6,000 IS WHERE A LOT OF OUR NOISE IS AND AROUND 6 6,000, THERE IS ONE AT EVERY MASS. WE WANT TED TO MAKE SHORURE THAT THERE WAS SOMETHING THAT WAS DECENT THERE. IT DOESN'T MEAN THE STUFF BELOW -- THEY'RE ALL REAL PEAKS, AS MY CHEMIST TELLS NE ME. SO WE WENT THROUGH THAT. SO WE DOWNSELECTED A LIST FROM THERE BECAUSE A LOT OF THE THREO THREONINES AND SERINES THAT WERE PRESENT WERE ALSO PRESENT ON THE SAME PEPTIDE, SO THAT ENDED UP REDUCING THE NUMBER OF PEPTIDE-A PEPTIDE-ADDUCT COMBINATION WE FOUND. THE CAV YET ON -- CAVEAT ON THIS IS THIS IS 32 OR MORE. I'M NOT SURE IF THE SAME PEPTIDE IS FOUND IN MULTIPLE PROTEINS. YOU CAN'T ALWAYS DIFFERENTIATE WHICH PROTEIN IS THERE BECAUSE THERE ARE MULTIPLE FORMS. SO THIS HAS BEEN -- THIS IS THE MINUIMAL LIST. THERE IS AT LEAST 32 DIFFERENT PROTEINS PRESENT. >> INAUDIB[INAUDIBLE] >> NOT WITH THE THREONINE. THERE IS ONE THAT I HAVE IN THE NEXT COUPLE OF SLIDES, WHERE I HAVE ONE PEPTIDE THAT HAD 2 TIYRO TYROSINE S S IN IT AND THEY ARE BOTH FAVID -- MODIFIED. IN THIS ONE HERE IT'S HIIMPOSSIBLE TO TELL. IF YOU END UP LOOKING HERE -- AT THE BEGINNING I HAD THIS EXAMPLE EXAMPLE. THIS IS THE ION SERIES AND THE TIYROSINE WASN'T IDENTIFIED. BUT THAT COULD HAVE BEEN THE RESULT OF A MODIFIED TIYROSINE BECAUSE IT'S THE DIFFERENT BETWEEN THE MASSES -- I'M SORRY LE. THE DIFFERENCE BETWEEN THE THREO THREONINE. IT COULD HAVE BEEN EITHER ONE. WE COULDN'T DIFFERENTIATE IT SO WE COULDN'T REPORT THE ONES THAT HAD TO BE ON THE TIYROSINE S. NOW, WE DIDN'T HAVE TO SAY IT WAS ON THE TIYROSINE. IF IT ONLY HAD ONE ADDUCT AND IT WAS REPORT ON THREONINE AND THAT PEPTIDE DID NOT HAVE A SER IINE OR TIYROSINE IN IT, WE DID NOT VERIFY THAT WE DEFINITE LLY HAD IT ON BOTH SIDES TO MAKE SHOURE. BUT -- SURE. BUT SINCE THESE ARE THE POTENTIAL TARGETS. ONE OF THE THINGS WE FEEL GOOD ABOUT IS THERE IS SOME INTERNAL VALID ATIATION. FIRST OF ALL, WHEN WE RAN UN UNTREATED SAMPLES, WE HAD NO A ADDUCTS IDENTIFIED. SO OUR FALSE POSITIVE RATE HERE IS BASED ON THAT WOULD BE 0%. THAT'S FOR THE ADDUCTS. THE POSITION MIGHT BE DIFFERENT. LOCATE, I CAN'T VERIFY. BUT FOR PEPTIDES HAVING ADDUCTS, EVERY ONE THAT WE HAD WAS PROBABLY REALLY AN ADDUCT SOME PLACE ON THAT PEPTIDE. BUT THEN FOR SOME OF THE LOCATIONS, WE ACTUALLY HAD VALID ATIONS WHERE WE HAD MULTIPLE THINGS HAPPENING, WHERE WE MIGHT HAVE HAD THE AGED AND UNAGED FORM ON THE SAME MASS. WE MIGHT HAVE HAD IN THIS CASE, THIS IS AN EXAMPLE WHERE WE HAD THE 2 TIYROSINES AND THIS PEPTIDE PEPTIDE. AND WE FOUND A PEPTIDE THAT ONLY THE FIRST WAS MODIFIED AND FOUND WHERE BOTH WERE MODIFIED AND ONLY THE LAUGHST ONE WAS MODIFIED. AND THEN WE ALSO DID IT WITH THE -- AND THIS IS THE CASE -- WHERE IN THIS PEPTIDE WE FOUND THE TIYROSINE, BOTH THE AGED AND UN UNAGED VERSIONS. SO THIS IS THE PEPTIDE WHERE WE HAD THE DIFFERENT LOCATIONS. SO IT'S HARD TO SEE HERE. THIS IS THE UNMODIFIED. THIS IS WHERE THE MODIFICATIONS ON THE 11TH. THIS IS WHERE IT'S ON THE 1ST AND THIS IS WHERE IT'S ON BOTH. NOW, THE CHALLENGE HERE IS SOME OF THESE PEPTIDES -- FROM THIS VIEW IT'S HARD TO SEE. BUT IF WE LOOK AT THE FIRST ONE, YOU CAN SEE THAT -- SO IF WE LOOK AT THIS SPECTRUM HERE AND YOU LOOK A LITTLE BIT TIGHTER, THERE IS ACTUALLY A PEAK, . SO THIS MASS DIFFERENCE BETWEEN THE 10TH AND 11TH ION SERIES IS RIGHT THE ADDITION AS A TIYROSINE PLUS A DIMETHYL GROUP ON IT. AND IF YOU LOOK AT THE MASS OF THE 11 HERE, WHICH IS THE UN UNMODIFIED ION VERSUS HERE, WE HAVE AN INCREASE OF 108 AND WE DO HAVE THIS TYPE OF SERIES IN THIS AS WELL. THE ONLY THING I WANT TO POINT OUT FU LOOK AT THESE, FU LOOK AT WHAT THE INTENSITY ARE, LIKE THIS IS THE 8TH AND 9TH AND 10TH SERIES, IT HAS THE SAME MAJOR PEAKS. SO WE BELIEVE THEY ARE THE SAME PEPTIDES, SOME OF THE CHARACTER CHARACTERISTICS MAKE IT LOOK LIKE THE SAME PEPTIDES. AND 10TH TO 11TH WE HAVE BOTH I IONES. THE OTHER ONES ARE FIRST ONES. IT'S REALLY IN THE BEGINNING. WE DON'T HAVE A STRONG SIGNAL THERE. WE'RE KIND OF AT THE LIMIT. THESE WERE DONE -- I DIDN'T MENTIONED THIS -- WHEN WE RUN THE INSTRUMENTS, WE USUALLY RUN THE FULL SCANS AT00,000 RES RESOLUTION AND THEN DOUGH WITH 10 HD DCHD AND WE HAVE A LITTLE BETTER COVERAGE. THESE ARE ACTUALLY DONE IN THE I ION TRAP. BUT WHAT WE'RE DOING IS WE'RE RIGHT AT THE BOTTOM END WHERE IT DINE ICALLY SETS THE RANGE OF THE INSTRUMENT. SO -- >> WHAT IS THE TYPE? >> IT IS CONCLUSION -- COLLISION-INDOIS -- COLLISION-IN COLLISION-INDUCE D ASSOCIATION. ARMY AKRCRONYMS. YEAH, THAT'S A PROBLEM WHEN YOU GET WORKING ON -- FOR AN ARMY. >> ONE OTHER THING. ALSO, ONE OF THE ISSUES IS WHEN YOU ARE DOINGxD THE ASSOCIATION OR THE HIGH-ENERGY ASSOCIATION, YOU CAN -- IT'S SOMETHING YOU COULD RUN. I HAD NO IDEA THIS COULD DO THAT THAT. THIS IS ANOTHER COMPLICATION TO BE AWARE OF. IT'S VERY TRICKY TO BE PRECISE. I MEAN, IF YOU ARE GETTING ENOUGH ENERGY -- >> RIGHT. I'LL KEEP THAT IN MIND. BUT WE DO HAVE -- THIS GOES BACK TO THE MODIFICATION. SO THESE PROTEINS ARE LIKE TARGETS. AND THIS REGION IS THE -- RIGHT. THE FACT THAT THESE LOCAL IIZE TO -- WE WOULDN'T HAVE IDENTIFIED THOSE IF THEY MOVED THE PTM. IF IT WASN'T ON A THREONINE AND A TIYROSINE, WE WOULD HAVE HAD IT FALSELY IDENTIFIED HERE. IF IT STARTED IN DIFFERENT AMINO ACID AND DURING THE COLLISION MOVED, THEN WE WOULD HAVE IS HAD A FALSE LE-POSITIVE THERE. WE DIDN'T GO THE OTHER WAY. SO THAT COULD HAVE BEEN SOME OF THAT IN OUR DATA AND WE'LL DEFINITELY LOOK FOR IT. ALL RIGHT. THIS IS THE OTHER EXAMPLE. THIS IS WHERE WE HAVE THE ADDUCT FOUND IN BOTH THE AGED AND UN UNAGED FORM. SO YOU SEE THE SERIES. THIS IS THE UNMODIFIED PEPTIDE, AND WE HAVE THE MESH SHIP FOR TIYROSINE. AND WE LOOK HERE AND WE HAVE THE 94 DALTONS, SO WE HAVE THE PROPER MASS SHIFT FOR THE TIYRO TYROSINE PLUS. AND HERE IS THE AGED FORM FROM THE -- WITH THE DIMETHYL GROUP, WHICH IS THE ORIGINAL. IF YOU WERE WONDERING, THESE ARE ACTUALLY TRIP LY CHARGED IONS THAT WE DID ON THIS SET. WE HAVE SOME OF THEM IN SINGLE CHARGED OR DOUBLY CHARGED AS WELL. I JUST SHOWED THESE AS AN EXAMPLE TODAY. ALL RIGHT, SO OVERALL WHAT DO WE FIND? WE FOUND 7 0B PROTEINS THAT HAD ADDUCT SITES. WE DON'T KNOW WHERE THE ADDUCT SITES WOULD BE BUT IT HAD A LOWER ABUNDANCE AS A RESULT OF THE TREATMENT WITH DICLOULOSIS BLOCKING THE BIOTIN. WE HAVE 32 PROTEINS THAT WE IDENTIFIED AND THESE ARE COLLECT DIRECT L KRCHC M S THAT HAVE THE A ADDUCT SITES. AND THIS IS THE SET THAT -- THESE PROTEINS, WHERE THERE COULD BE SOME QUESTION ON WHETHER THE SITE IS DEAD ON ON THAT LOCATION, BUT THEY ARE VERY GOOD TARGETS. BUT IN 10 OF THESE PROTEINS, -- ACTUALLY, WE'RE ONLY OFF BY 10 PROTEINS -- 3 OF THESE ARE IN THE GLY COL SISIS PATHWAY. SO WHAT MAKES THIS INTERESTING IS THAT ONE OF THE POTENTIAL MECHANISMS THAT IS DRIVING -- THAT HAS BEEN POSED AS A SECOND ARY TARGET OR THE OFF-TARGET EFFECTS OF THAT HAVE TO DO WITH ENERGY METABOLISM IN THE MIGTO MITOCONDRI IA. SO THE FACT THAT WE'RE MODIFYING THEM IS OF POTENTIAL INTEREST. WE DID LOOK TO SEE IF THERE WAS ANY SORT OF SPEC FIIFICITY THAT WE ARE BUYING INTO. WE DIDN'T SEE ANYTHING SPECIFICALLY OF THE SEQUENCE. KIND OF OUR GUT FEELING IS THAT IT'S SURFACE PROTEINS THAT ARE AVAILABLE. THESE HIGH CONCENTRATION S ARE PROBABLY WHAT WE'RE TARGETING. WE DID NOT FIND THAT MANY SER INE TARGETS. LESS THAN 20% OF THE TOTAL AND MOST OF THE TARGETS WERE TIYRO TYROSINES, WHICH IS INTERESTING BECAUSE SERINE IS THE ACTOSITE. AS PREVIOUSLY REPORTED BY OTHER GROUPS, THE MAJORITY OF THE A ADDUCTS WERE WITHIN THE 8 AMINO ACIDS AND IN OUR CASE, ALL OF THE ADDUCTS WERE WITHIN 8 OF THE AMINO ACIDS. NOT CLEAR WHETHER THAT IS A BIAS DUE TO THE FACT THAT THE PEP PEPTIDES. IT WAS SIGNIFICANT USING THE A AMINO ACID BIAS IN THE PEPTIDES TO IDENTIFY. BUT IT STILL COULD BE -- THE FACT THAT ALL OF THEM WERE THERE MAKES IT INTERESTING. A FAIR NUMBER OF THE SITES ARE ACTUALLY BIOLOGICAL LY RELEVANT "" AS FOR PHORYLATION SITES AND MET METABINDING SITES. SO PUTTING AN ADDUCT ON THESE SITES DEFINITELY HAS THE POTENTIAL TO INTERFERE WITH BIOLOGICAL ACTIVITY OF THESE PRO PROTEINS. NOW, IN TERMS OF SPEC FIS.IFICITY, TURNS OUT THAT ONE UT OF EVERY PEP THEID WE IDENTIFIED HAD AN A ADEDUCT ON IT. SO IF WE START DOING THE MATH BACKWARDS, THAT'S KIND OF ABOUT 1 PEPTIDE PER PROTEIN ON AVERAGE. THAT MEANS THAT THERE IS NOT A LOT OF SPEC FIZIFICITY AT THESE HIGH CONCENTRATIONS. IT'S A REACT IIVE CHEMICAL. WE'RE TRYING TO IDENTIFY SPEC FIS ITY. AND BECAUSE WE DID AT CERTAIN CONCENTRATIONS, SOME OF THEM WERE IDENTIFIED AT LOWER CON CONCENTRATIONS AS WELL TO TRY TO DO SOME SORT OF OF QUANTITY QUANTITIFICATION OF HOW THEY CHANGE TO GET US MORE INFORMATION ABOUT THE SPEC FIZZ SPECIFICITY WITH THE DIFFERENT SITES. SO, BECAUSE THE BIOLOGICAL IMP IMPLICATIONS OF THE ENERGY MET METABOLISM IS THE POTENTIAL CAUSE OF SOME OF THE;– TARGET EFFECTS OF DICHLOROIS EXPOSURES, WE DECIDED TO PUT 2 OF THE EN ENZYMES OF THE -- LDH AND ALSO, THE ACTIVITIES ARE COMMERCIALLY AVAILABLE AND THE PROTEINS ARE COMMERCIALLY AVAILABLE, WHICH MAKES IT REALLY EASY TO DO THESE EXPERIMENTS. AND IN THIS CASE HERE, BOTH PRO PROTEINS WERE IDENTIFIED IN THE FT BIOTIN ASSAY, BUT THE LDH WAS NOT -- WE DIDN'T IDENTIFY ANY A ADDUCTS ON IT WITH THE DIRECT A ASSAY. IT JUST MEANS WHEN WE'RE LOOKING AT WHOLE CELLS, WE MIGHT NOT SEE IT. SO WE THOUGHT IT HELPS IDENTIFY THE BIOTIN WITH MUCH MORE CON CONCENTRATIONS THAN THE PEPTIDES PEPTIDES. AND IF WE HAVE A BETTER CHANCE OF FINDING THE MODIFICATIONS. WE PERFORM THE ENSDIEMENT IC A ASSAYS BOTH USING PURIFIED PRO PROTEINS AND DOING -- ENZYMAT IC SA ASSAYS BOTH USING THE PURIFIED PROTEINS. WE DIDN'T DO TREATED EXTRACTS USING CELL LINES. WE ALSO DO -- WE ALSO DID THE DIRECT ASSAY OF THE PROTEIN DI DICHLOROIS TO MAKE SHOURE WE GET A BETTER CHANCE TO LOOK AT THEM. SO THE ENZYMAT IC APROTEINS. WHAT WE FOUND WAS THE ACTIVITY WAS REDUCED BOTH WITH THE PURIFIED LDH AND THE LIG AASE WITH THE TREATED CELL LINES. BUT THE LD SHELTH WAS NOT SIGNIFICANTLY AFFECTED. IT DEFINITELY APPEARS TO BE A TARGET. WE'RE SEEING THE MODIFICATIONS. IF WE LOOK AT THE ADDUCTS, WE FOUND A COUPLE MORE SITES WITH POTENTIAL MODIFICATIONS WITH IMPORTANTLY BIOLOGICAL ROLES. ONE OF THE SITES IDENTIFIED IS THE ACTIVE SITE FOR NDH BINDING. ANOTHER IS THE FOSPHOSPHORYLATION. SO WE THINK IT'S A VERY GOOD TARGET FOR FOLLOWUP STUDIES FOR POTENTIAL DICLOFB OOSE OSOSE WHAT DOES THIS SAY ABOUT OUR FT BIOTIN? IT SAYS THAT IT'S PROBABLY AN EN ENRICHED THING, BUT IT MIGHT OR MIGHT NOT BE EVERY PROTEIN IN THERE. AND IT DOESN'T MEAN THERE IS AN ADEDUCT ON THE LDH. WE DONE SEE IT WHEN WE DO ASSAY. WE DO NOT SEE IT ON THE INSTRUMENT. IT DOESN'T MEAN IT'S NOT THERE. SO IT'S IDENTIFIED BY LCM. AND 2 OF THEM HAVE BIOLOGICAL ROLES SO THIS IS DEFINITELY A GOOD TARGET TO MOVE FORWARD WITH WITH. SO IN ADDITION TO JUST LOOKING AT THESE SITES, WE WANTED TO TRY TO GET A BETTER FEEL OF THE BIOLOGY, TAKING INTO ACCOUNT ALSO THE NEW ADDUCTS THAT WE'VE FOUND. SO WE DID A ROUND OF QUANTIUM PROTEOMICS, WHERE WE EXPOSED THE CELL LINES TO MICRO MORAL FOR 4 OR 24 HOURS. SO WE DID 2 DIFFERENT TIME POINTS. THESE ARE HUMAN LIVER CELLS. WHICH IS ANOTHER THING, IF YOU THINK ABOUT IT. TRADITIONAL TARGET OF DIKROEFB OSE IS NORMAL, BUT WE DO A LOT OF WORK IN LIVER. THE IDEA OF BIOMARKER DISCOVERY, THE FACT THAT THE LIVER HAS SO MUCH INTERACTION WITH BLOOD AND IS THE PRIMARY ORGANIZ -- PRIMARY ORGANI FOR PROCESSING CHEMICALS, THAT WE HOPE IT'S GOING TO BE RICH FOR FINDING BIOMARKERS. THAT'S WHY ALL OF OUR WORK IS BASED ON THE LIVER. AND THAT WAS ONE OF THE REASONS. WE HAVE ANOTHER STUDY, WHICH IS IN THE TRANSCRIPTS BEING PREPARED, WHERE WE'VE DONE RODE RODENT SIZE AND LOOKED AT BRAIN TRANSCRIPTS AND CELL CHANGES. BUT THE PERSON -- THE ACTUALLY FROM THAT STUDY, WE ACTUALLY DID SOME LIVER WORK AS WELL, BUT WE DIDN'T SEE ANYTHING THAT CAUSED THE LOW-LEVEL NEW JERSEY ICICAL EFFECTS -- THE LOW-LEVEL NEWER LODGE ICAL EFFECTS WERE NOT CAUSING THIS. WE DID -- AND WAS GOING TO DO AN ANALGUS STUDY AND ENDED UP MOVING ON BEFORE WE GOT TO IT. IN THIS CASE, WE DID THE CELL LINE USING WE DID A SLIGHTLY DIFFERENT THING HERE. WE DID INCREASE THE STATISTICS. FOR EXAMPLE, WE ENDED UP EXPORT EXPORTING THE PEPTIDES FROM PRO GENESIS AND USING ANOTHER SOFTWARE PACKAGE WE HAVE TO DO STATISTICS ON THAT SO THE PEP PEPTIDES CAN BE CHANGING. AND PUT BACK THE PROTEINS, WE USED THE PROTEINS THAT WERE CHANGING BY PRO GENESIS. SO WE DID STATISTICS ON THE PEP PEPTIDE. SO WE HAD A PEPTIDE THAT WAS CHANGING BY 1 AT 5 SIGNIFICANTLY STATISTICALLY, AND THEN THE PRO PROTEIN HAD TO BE CHANGING BY 1. 1.5 ALSO. SO WE IDENTIFIED PROTEINS CHANGING IN ABUNDANCE. 21 OF THEM AT 4 HOURS AND 20 AT 24 HOURS AND ONLY 16 OF THEM OVERLAPPED. DURING THIS EXPERIMENT, WE ALSO LOOKED FOR MODIFICATIONS USING THE, BASED ON THE ADDUCTS, IN THE DATABASE SEARCHES. BUT WE ACTUALLY DID SEE IT. FROM THAT LIST, WE USED PATH WWAY ANALYSIS CALLED MET ACORE FROM GENE GILL AND WE IDENTIFIED 27 PATHWAYS THAT HAD AT LEAST ONE OF THE PROTEINS THAT WERE CHANGING IN ABUNDANCE AND ONE OF THE DVD PROTEINS. SO ONE OF THE PROTEINS WE IDENTIFIED IN ONE OF OUR TWO A ASSAYS IN THE BEGINNING. AND FROM THAT, WE BASICALLY IDENTIFIED 6 BIOLOGICAL PROCESS THAT'S WERE -- PROCESS THAT'S WERE CHARGED BY OVEREXPOSURE. FU WOULD LIKE TO LOOK AT THE TABLES, THESE ARE THE PROCESSES AND THE MEDICAL MAPS THAT WERE AFFECTED. THE BIG ONES, I THINK, ARE IMPORTANT TO SURVIVAL, WHICH I THOUGHT WIDELY THERE IS A DIFFERENCE IN PRO TEENS BETWEEN THE 4 HOURS AND 24 HOURS AND A CHANGE IN STATUS AND CELLS. AND REGULATION IS IMPORTANT. THE GLUCOSE ENZYMES WHERE WE LOOKED AT AND METABOLISM. OUR DATA SUPPORTS THAT. SO WE ALSO SAW THE CHANGE IN THE PROTEINS IN THAT AT 4 HOURS, CELLS LOOKED TO BE MORE AT AN AH APOP TTOTIC STATE. THE ONLY DIFFERENCE IS WE'RE SEEING IN THE PROTEINS BETWEEN THOSE TWO LEVELS. BUT THE FACT THAT WE'VE IDENTIFIED THIS LARGE NUMBER OF PROTEINS -- ANY BIOLOGICAL ROBUST PROTEINS HAVE HAS THE POTENTIAL TO BE PERTURBED. THESE ARE HIGH CONCENTRATIONS, BUT THE REACTIVITY OF THE MOLL MOLECULE CAN DETERMINE A LOT OF DIFFERENT THINGS IN THE CELLS. SO WE'VE IDENTIFIED 70 DIFFERENT CANDIDATES. 51 DIFFERENT ADDUCT COMBINATIONS COMBINATIONS. WE SHOWED THAT ENZYMAT IC ACTIVITY IS ACTUALLY REDUCED BY DICHLOROIS AND IS AN INHIBITOR AS WELL. WE HAVE 49 PROTEINS WHOSE ABUNDANCE CHANGES WITH TREATMENTS, THAT WE CAN THEN START TO GET INTO SOME OF THE BIOLOGY AND ENERGY AND MET METABOLISM BEING THE ONE THAT WE'LL FOLLOW UP SCAAND WHERE I WOULD FOCUS MY ENERGY. SO, AS I POINTED OUT, THESE WERE HIGH CONCENTRATIONS OF DICHLORO DICHLOROIS. IF WE REALLY WANT TED TO VALIDATE THESE, WE REALLY NEED TO GET DOWN INTO LOWER LEVELS OF SENSE SENSITIVITY. SO PROBABLY THROUGH TARGET A APPROACHES. SO WITH THE T Q, WE CAN DO R M (M)RNA ANALYSIS, OR WE CAN DO PURIFICATION VALUES TO IDENTIFY THE PROTEINS WE ARE INTERESTED AND DO ANALYSIS OF JUST THOSE PROTEINS. ANOTHER THING IS WE DIDN'T IDENTIFY ALL AT DUCTS THAT WE IDENTIFIED DIRECTLY WERE IN TREATED CELL EXTRACTS, NOT IN TREATED CELL LINES. SO THE BIOTIN WAS NOT DONE WITH CELL LINES, SO THAT MIGHT HELP US TO GET AT THE LOWER-ABUNDANT MODIFICATIONS. AND WE CAN ALSO DO THE TARGET ANALYSES IN THE CELL LINES, OF THE SOME OF THE PROTEINS IDENTIFIED. THAT'S PRETTY MUCH IT. ANYBODY HAVE ANY QUESTIONS? APPLAU [APPLAUSE] >> SO YOUR INITIAL EXPERIMENT TREATED CELL, RIGHT? SO BIOLOGICAL LY -- AND -- WOULD YOU THINK THAT IT WAS MODIFIED MEMBRANES MOST LIKELY AFFECTING THE ENVIRONMENTS THAT YOU HAD MODIFICATION THROUGH RECEPTORS, RATHER THAN IT ACTUALLY ENTERING THE CELL? OR DOES IT DELIVER CELLS IN IN MANNER? I DON'T KNOW. >> ACTUALLY I'M NOT FAMILIAR WITH THE UPTAKE EFFECT, WHICH IS AN INTERESTING QUESTION. BECAUSE WE WHEN WE DO THE CELL EXTRACTS, WE DID CELLULAR PRO PROTEINS. WE DID NOT DO MEMBRANE. >> AND IT MIGHT BE WHY THE LIVER IN THE CELLS BUT THE -- >> WE DID SEE A REDUCTION IN GAT H ACTIVITY IN TREATED CELLS, THOUGH,. SO IT IS AFFECTING INSIDE OF THE CELL. RIGHT. >> BUT FLAU[INAUDIBLE] >> IT COULD BE THE RECEPTOR AS WELL. BUT SOME OF THE AFFECTS -- EFFECTS SUGGESTED THAT IT MIGHT BE GETTING INTO THE CELL. IT'S PROBABLY AT A LOWER CON CONCENTRATION THAN IT IS. AND YOU REMEMBER LIVER CELLS ARE DETOXIFIED CELLS. THEY LIKE TO TAKE UP THINGS AND GET RID OF THEM. SO THE LIVER CELLS ARE THE BREAK BREAKDOWN OF DICHLOROOSE AND IT IS IN LIVER. SO IT IS BEING UPDATED. THE CONCENTRATION ON THE MEM MEMBRANE PROTEINS COULD VERY MUCH BE HIGHER AND THAT COULD BE THE TARGETS ALSO OF LOOKING FOR EFFECTS. APPLAU[APPLAUSE]