1 00:00:42,669 --> 00:00:44,538 SEMINAR CONTRIBUTIONS TO THE 2 00:00:44,538 --> 00:00:46,073 UNDERSTANDING OF IMMUNE CELL 3 00:00:46,073 --> 00:00:48,041 SIGNALING AND PROGRAMMED CELL 4 00:00:48,041 --> 00:00:51,445 DEATH, NOT GOING INTO MANY 5 00:00:51,445 --> 00:00:56,149 DISCOVERIES BUT JUST HIGHLIGHT 6 00:00:56,149 --> 00:00:57,451 TWO EXAMPLES, HE DISCOVERED 7 00:00:57,451 --> 00:01:03,056 CASPASE 3 AND ITS INVOLVEMENT IN 8 00:01:03,056 --> 00:01:05,125 THE APOPTOSIS DOWN STREAM OF 9 00:01:05,125 --> 00:01:07,027 RECEPTORS AND MORE RECENTLY 10 00:01:07,027 --> 00:01:13,867 DISCOVERED THE PATHWAY OF 11 00:01:13,867 --> 00:01:14,701 NON-CANONICAL PYROPTOSIS. 12 00:01:14,701 --> 00:01:16,236 THANK YOU FOR COMING, WE'RE 13 00:01:16,236 --> 00:01:26,646 LOOKING FORWARD TO YOUR TALK. 14 00:01:26,646 --> 00:01:37,224 >> THANKS FOR ACCOMMODATING ME. 15 00:01:37,424 --> 00:01:42,162 IT'S AN UNUSUAL HOUR. 16 00:01:42,162 --> 00:01:47,067 WHAT I THOUGHT I WOULD DO TODAY 17 00:01:47,067 --> 00:01:51,238 IS DISCUSS SOME RELATIVELY 18 00:01:51,238 --> 00:01:52,906 RECENT WORK ON FORMATION AND 19 00:01:52,906 --> 00:01:55,342 MEMBRANES, HOW THAT RELATES TO 20 00:01:55,342 --> 00:01:55,942 INNATE IMMUNE MECHANISMS. 21 00:01:55,942 --> 00:01:59,479 I'LL GIVE YOU A BIT OF A 22 00:01:59,479 --> 00:02:00,781 HISTORICAL CONTEXT AND THEN I'LL 23 00:02:00,781 --> 00:02:04,284 FINISH UP WITH SOME MORE RECENT 24 00:02:04,284 --> 00:02:04,918 DATA. 25 00:02:04,918 --> 00:02:06,453 SO, THE FIRST QUESTION I'D LIKE 26 00:02:06,453 --> 00:02:08,755 TO ADDRESS IS THIS QUESTION THAT 27 00:02:08,755 --> 00:02:13,059 HAS LOOMED LARGE IN CELL BIOLOGY 28 00:02:13,059 --> 00:02:14,461 AND IMMUNOLOGY FOR DECADES 29 00:02:14,461 --> 00:02:18,298 REALLY, WHICH IS HOW A 30 00:02:18,298 --> 00:02:19,499 LEADERLESS CYTOKINE, CYTOKINES 31 00:02:19,499 --> 00:02:23,770 THAT LACK A SIGNAL PEPTIDE, 32 00:02:23,770 --> 00:02:26,506 PROTOTYPICAL EXAMPLES BEING IL-1 33 00:02:26,506 --> 00:02:28,975 BETA AND IL-18, HOW ARE THEY 34 00:02:28,975 --> 00:02:29,242 RELEASED? 35 00:02:29,242 --> 00:02:30,811 SO, BEFORE I GET TO THIS 36 00:02:30,811 --> 00:02:35,649 QUESTION, I NEED TO GIVE YOU A 37 00:02:35,649 --> 00:02:38,051 BACK GROUND ON IL-1 BETA AND 38 00:02:38,051 --> 00:02:41,321 IL-18, JUST TO REMIND YOU THESE 39 00:02:41,321 --> 00:02:45,392 CYTOKINES DISCOVERED IN THE 40 00:02:45,392 --> 00:02:47,661 '80s WERE INITIALLY IDENTIFIED 41 00:02:47,661 --> 00:02:52,365 AS EXISTING IN A SENSE IN A PRO 42 00:02:52,365 --> 00:02:54,568 FORM WHERE THEY WERE 43 00:02:54,568 --> 00:02:57,737 BIOLOGICALLY INERT, AND THEY HAD 44 00:02:57,737 --> 00:03:01,241 TO BE PROTEOLYTICALLY PROCESSED 45 00:03:01,241 --> 00:03:03,109 TO GIVE YOU MATURE CYTOKINE IL-1 46 00:03:03,109 --> 00:03:03,877 AND IL-18. 47 00:03:03,877 --> 00:03:08,381 IT WAS REASONED IF ONE WAS ABLE 48 00:03:08,381 --> 00:03:09,883 TO IDENTIFY PROTEASE RESPONSIBLE 49 00:03:09,883 --> 00:03:12,185 FOR THIS PROCESSING AND INHIBIT 50 00:03:12,185 --> 00:03:13,620 THAT PROTEASE, THEN YOU WOULD 51 00:03:13,620 --> 00:03:15,689 FREEZE THE CYTOKINES IN THIS 52 00:03:15,689 --> 00:03:18,992 INACTIVE PRO FORM, THAT WOULD BE 53 00:03:18,992 --> 00:03:20,126 BE A EFFECTIVE 54 00:03:20,126 --> 00:03:20,527 ANTI-INFLAMMATORY. 55 00:03:20,527 --> 00:03:24,464 AND SO THE RACE WAS ON TO 56 00:03:24,464 --> 00:03:25,232 IDENTIFY THIS PROTEASE, AND IT 57 00:03:25,232 --> 00:03:32,138 TURNED OUT TO BE A MOST UNUSUAL 58 00:03:32,138 --> 00:03:37,210 PROTEASE, INTERLEUKIN 1 59 00:03:37,210 --> 00:03:43,316 CONVERTING ENZYME CLONED BY 60 00:03:43,316 --> 00:03:47,487 BLACK AND THORNBERRY. 61 00:03:47,487 --> 00:03:51,424 IT CLEAVED ACID RESIDUE, VERY 62 00:03:51,424 --> 00:03:54,594 UNUSUAL SUBSTRATE SPECIFICITY. 63 00:03:54,594 --> 00:03:57,664 CYSTEINE PROTEASE, BUT UNUSUAL 64 00:03:57,664 --> 00:03:59,866 SUBSTRATE SPECIFICITY. 65 00:03:59,866 --> 00:04:01,368 UNBEKNOWNST TO THEM THERE WAS A 66 00:04:01,368 --> 00:04:04,371 RACE ON TO IDENTIFY GENES 67 00:04:04,371 --> 00:04:09,209 RESPONSIBLE FOR CELL DEATH, IN 68 00:04:09,209 --> 00:04:11,044 THE DEVELOPMENTAL ORGANISM C. 69 00:04:11,044 --> 00:04:11,478 ELEGANS. 70 00:04:11,478 --> 00:04:18,184 THEY HAD IDENTIFY THIS PROTEASE 71 00:04:18,184 --> 00:04:28,595 THAT ALSO EXISTS IN THE XYMOGEN 72 00:04:28,595 --> 00:04:28,795 FORM. 73 00:04:28,795 --> 00:04:30,664 AS I MENTIONED, THEY WERE 74 00:04:30,664 --> 00:04:32,866 UNAWARE THERE WAS AN INTEREST IN 75 00:04:32,866 --> 00:04:36,570 CELL DEATH IN THE WORM, YOU 76 00:04:36,570 --> 00:04:37,771 COULDN'T BE FURTHER AFIELD FROM 77 00:04:37,771 --> 00:04:40,273 WHAT THEY WERE THINKING ABOUT. 78 00:04:40,273 --> 00:04:43,376 AND YET THIS DISCOVERY OF 79 00:04:43,376 --> 00:04:47,080 INTERLEUKIN 1 CONVERTING ENZYME 80 00:04:47,080 --> 00:04:48,248 WAS FUNDAMENTAL IMPORTANCE 81 00:04:48,248 --> 00:04:52,652 BECAUSE WITH IT CAME REALIZATION 82 00:04:52,652 --> 00:04:57,290 THAT THE CENTRAL EXECUTOR OF 83 00:04:57,290 --> 00:05:00,226 CELL DEATH THAT PREVIOUSLY HAD 84 00:05:00,226 --> 00:05:01,962 NO KNOWN FUNCTION, WAS A 85 00:05:01,962 --> 00:05:06,333 MYSTERIOUS GENE IN THE WORM, WAS 86 00:05:06,333 --> 00:05:08,368 ACTUALLY -- HAD HOMOLOGY TO 87 00:05:08,368 --> 00:05:11,204 INTERLEUKIN 1 BETA CONVERTING 88 00:05:11,204 --> 00:05:13,173 ENZYME. 89 00:05:13,173 --> 00:05:19,279 CED-3 WAS A PROTEASE, A DEATH 90 00:05:19,279 --> 00:05:22,148 PROTEASE, THAT CLEAVED ACID 91 00:05:22,148 --> 00:05:22,582 RESIDUE. 92 00:05:22,582 --> 00:05:27,053 THIS IS A GREAT EXAMPLE HOW 93 00:05:27,053 --> 00:05:28,788 SEEMINGLY UNRELATED AREAS OF 94 00:05:28,788 --> 00:05:31,224 RESEARCH CAN COME TOGETHER AND 95 00:05:31,224 --> 00:05:37,364 ILLUMINATE A WHOLE NEW HIGHWAY 96 00:05:37,364 --> 00:05:38,098 OF DISCOVERY. 97 00:05:38,098 --> 00:05:41,067 SINCE THEN LOT OF CASPASES HAVE 98 00:05:41,067 --> 00:05:41,735 BEEN IDENTIFIED. 99 00:05:41,735 --> 00:05:46,339 THEY ARE A FAMILY NOW, MAMMALIAN 100 00:05:46,339 --> 00:05:46,640 CASPASES. 101 00:05:46,640 --> 00:05:48,074 AND YOU'RE PROBABLY FAMILIAR 102 00:05:48,074 --> 00:05:50,710 WITH THE ONES THAT PLAY A 103 00:05:50,710 --> 00:05:52,879 PROMINENT ROLE IN APOPTOSIS SUCH 104 00:05:52,879 --> 00:05:57,817 AS CASPASE 3 AND CASPASE 7. 105 00:05:57,817 --> 00:06:02,555 BUT THE GRANDDADDY OF THEM ALL, 106 00:06:02,555 --> 00:06:04,524 OF COURSE, IS INTERLEUKIN 1 107 00:06:04,524 --> 00:06:06,393 CONVERTING ENZYME, CASPASE 1, 108 00:06:06,393 --> 00:06:09,029 THAT'S HOW IT ALL STARTED. 109 00:06:09,029 --> 00:06:12,832 AND CASPASE 1 PLAYS A PROMINENT 110 00:06:12,832 --> 00:06:14,601 ROLE IN INFLAMMATION, AND THERE 111 00:06:14,601 --> 00:06:18,405 ARE OTHER CASPASES THAT DO SO AS 112 00:06:18,405 --> 00:06:18,838 WELL. 113 00:06:18,838 --> 00:06:21,608 AND THESE ARE CASPASE-4 AND 5 IN 114 00:06:21,608 --> 00:06:24,210 THE HUMAN REPRESENTED AS A 115 00:06:24,210 --> 00:06:26,312 SINGLE CASPASE, CASPASE-11 IN 116 00:06:26,312 --> 00:06:28,815 THE MOUSE, AND THIS -- THESE ARE 117 00:06:28,815 --> 00:06:33,219 CASPASES THAT FUNDAMENTALLY ARE 118 00:06:33,219 --> 00:06:36,056 IMPORTANT IN NON-CANONICAL 119 00:06:36,056 --> 00:06:38,792 INFLAMMASOME PATHWAY. 120 00:06:38,792 --> 00:06:40,760 SO, AGAIN, I'LL EMPHASIZE THESE 121 00:06:40,760 --> 00:06:45,165 ARE CYSTEINE PROTEASES, AND THEY 122 00:06:45,165 --> 00:06:46,700 ALL CLEAVE ACID RESIDUE. 123 00:06:46,700 --> 00:06:50,070 CASPASE 1 IS THE CASPASE THAT IS 124 00:06:50,070 --> 00:06:52,038 RESPONSIBLE FOR THE PROTEOLYTIC 125 00:06:52,038 --> 00:06:58,044 MATURATION OF IL-1 BETA AND 126 00:06:58,044 --> 00:06:58,244 IL-18. 127 00:06:58,244 --> 00:07:01,981 I NEED TO INTRODUCE YOU TO THE 128 00:07:01,981 --> 00:07:02,782 INFLAMMASOME, SO THE 129 00:07:02,782 --> 00:07:06,152 INFLAMMASOME CAN BE THOUGHT OF 130 00:07:06,152 --> 00:07:13,426 AS A CYTOSOLIC APPARATUS THAT IS 131 00:07:13,426 --> 00:07:16,162 RESIDENT IN THE CYTOPLASMIC 132 00:07:16,162 --> 00:07:17,931 COMPARTMENT OF INNATE IMMUNE 133 00:07:17,931 --> 00:07:28,475 CELLS, INCLUDES END THEY'LLAL 134 00:07:29,275 --> 00:07:33,513 CHYLAL -- EPITHELIAL CELLS. 135 00:07:33,513 --> 00:07:36,249 IT RESPONDS TO VIOLATIONS BY 136 00:07:36,249 --> 00:07:38,418 ACTIVATING INNATE IMMUNE SYSTEM, 137 00:07:38,418 --> 00:07:40,954 IN THIS CASE HERALDED BY THE 138 00:07:40,954 --> 00:07:43,490 GENERATION OF MATURE IL-1 BETA 139 00:07:43,490 --> 00:07:44,257 AND IL-18. 140 00:07:44,257 --> 00:07:46,326 SO, IT'S A COMPLEX TOPIC, AND I 141 00:07:46,326 --> 00:07:49,529 WILL AT THE RISK OF 142 00:07:49,529 --> 00:07:50,430 OVERSIMPLIFICATION, BECAUSE IT 143 00:07:50,430 --> 00:07:52,799 ISN'T THAT BUT THE REST OF MY 144 00:07:52,799 --> 00:07:54,667 TALK I'LL JUST REMIND PEOPLE 145 00:07:54,667 --> 00:07:58,037 THAT THERE'S A NON-CANONICAL 146 00:07:58,037 --> 00:07:59,272 PATHWAY THAT RESPONDS TO 147 00:07:59,272 --> 00:08:04,210 INTRACELLULAR LPS AND ENGAGES 148 00:08:04,210 --> 00:08:07,280 CASPASE-11 AND CANONICAL PATHWAY 149 00:08:07,280 --> 00:08:09,883 THAT RESPONDS TO BEWILDERING 150 00:08:09,883 --> 00:08:14,988 ARRAY OF PAMPS AND DAMPS, 151 00:08:14,988 --> 00:08:17,357 TOXINS, ENGAGES CASPASE 1. 152 00:08:17,357 --> 00:08:21,494 AND CASPASE-11 IN FACT 153 00:08:21,494 --> 00:08:23,263 COMMUNICATES WITH CASPASE 1, 154 00:08:23,263 --> 00:08:25,231 THIS CONVERTING ENZYME IS 155 00:08:25,231 --> 00:08:28,301 RESPONSIBLE FOR THE PROTEOLYTIC 156 00:08:28,301 --> 00:08:30,804 MATURATION OF THESE CYTOKINES. 157 00:08:30,804 --> 00:08:34,641 I WANTS TO MAKE AN IMPORTANT 158 00:08:34,641 --> 00:08:36,476 POINT EVEN AFTER PROTEOLYTIC 159 00:08:36,476 --> 00:08:38,878 MATURATION THESE CYTOKINES LACK 160 00:08:38,878 --> 00:08:42,081 A SIGNAL PEPTIDE. 161 00:08:42,081 --> 00:08:44,717 SO, THERE'S NO ER GOLGI PATH FOR 162 00:08:44,717 --> 00:08:46,019 THEIR RELEASE. 163 00:08:46,019 --> 00:08:47,453 HOW ARE THEY GETTING OUT? 164 00:08:47,453 --> 00:08:51,391 AND WHAT ONE REALIZED IN ABOUT 165 00:08:51,391 --> 00:08:55,094 2015 WAS THAT BOTH CASPASE-11 166 00:08:55,094 --> 00:08:57,263 AND CASPASE-1, ONCE ACTIVATED, 167 00:08:57,263 --> 00:09:00,567 CAN ENGAGE A FORM OF NECROTIC 168 00:09:00,567 --> 00:09:03,837 DEATH TERMED PYROPTOSIS THAT IS 169 00:09:03,837 --> 00:09:05,905 CHARACTERIZED BY INTENSE 170 00:09:05,905 --> 00:09:06,539 MEMBRANE PERFORATION. 171 00:09:06,539 --> 00:09:09,742 AND IT WAS PRESUMED THAT THIS 172 00:09:09,742 --> 00:09:10,844 MEMBRANE PERFORATION PROVIDES 173 00:09:10,844 --> 00:09:16,449 THE CHANNELS FOR THE EXIT OF 174 00:09:16,449 --> 00:09:19,919 THESE MATURE CYTOKINES. 175 00:09:19,919 --> 00:09:24,958 SO THE RELEASE OF LEADERLESS 176 00:09:24,958 --> 00:09:26,726 CYTOKINES WAS THROUGH A 177 00:09:26,726 --> 00:09:29,462 MECHANISM THAT INVOLVED MEMBRANE 178 00:09:29,462 --> 00:09:30,997 PERFORATION, AND THEIR ACCESS TO 179 00:09:30,997 --> 00:09:33,733 THE INTRACELLULAR MILIEU. 180 00:09:33,733 --> 00:09:37,237 181 00:09:37,237 --> 00:09:39,405 ANOTHER QUESTION WAS, WELL, HOW 182 00:09:39,405 --> 00:09:41,908 ARE THE MEMBRANES GETTING 183 00:09:41,908 --> 00:09:42,408 PERFORATED? 184 00:09:42,408 --> 00:09:45,545 WELL, IN ORDER TO ADDRESS THAT, 185 00:09:45,545 --> 00:09:47,647 WE CARRIED OUT A FORWARD GENETIC 186 00:09:47,647 --> 00:09:50,583 SCREEN. 187 00:09:50,583 --> 00:09:53,753 THIS IS PUBLISHED SO I'LL GO 188 00:09:53,753 --> 00:09:59,459 OVER IT QUICKLY. 189 00:09:59,459 --> 00:10:01,194 THIS INDUCES POINTS MUTATIONS, 190 00:10:01,194 --> 00:10:03,263 IT'S A VERY LARGE PROGRAM. 191 00:10:03,263 --> 00:10:06,666 TENS OF THOUSANDS OF PEDIGREES 192 00:10:06,666 --> 00:10:07,200 WERE GENERATED. 193 00:10:07,200 --> 00:10:10,036 AND THE QUESTION WAS SIMPLE. 194 00:10:10,036 --> 00:10:13,139 WE ISOLATED MACROPHAGES FROM 195 00:10:13,139 --> 00:10:15,008 THESE MUTAGENIZED MICE AND ASKED 196 00:10:15,008 --> 00:10:19,145 IF THEIR ABILITY TO RELEASE IL-1 197 00:10:19,145 --> 00:10:20,146 BETA WAS COMPROMISED. 198 00:10:20,146 --> 00:10:21,648 THIS IS ONE EXAMPLE. 199 00:10:21,648 --> 00:10:23,583 A FEW HUNDRED MICE HERE. 200 00:10:23,583 --> 00:10:26,619 YOU STIMULATE THEM WITH 201 00:10:26,619 --> 00:10:30,023 CYTOSOLIC LPS, THEY MAKE COPIOUS 202 00:10:30,023 --> 00:10:33,092 BUCKET LOADS OF IL-1 BETA THAT'S 203 00:10:33,092 --> 00:10:33,626 RELEASED. 204 00:10:33,626 --> 00:10:41,401 NOW, IN RESPONSE TO LPS 205 00:10:41,401 --> 00:10:45,538 CASPASE-11 MUTANT DOESN'T MAKE 206 00:10:45,538 --> 00:10:46,239 IL-BETA. 207 00:10:46,239 --> 00:10:48,841 CASPASE-11 WAS DOWNSTREAM OF 208 00:10:48,841 --> 00:10:49,208 LPS. 209 00:10:49,208 --> 00:10:52,578 BUT WHAT CAUGHT OUR ATTENTION 210 00:10:52,578 --> 00:10:58,885 WAS ANOTHER MUTANT STRAIN B1 1, 211 00:10:58,885 --> 00:11:03,156 AND AT THIS JUNCTURE WE WERE 212 00:11:03,156 --> 00:11:07,994 QUITE SURPRISED, WE NEVER HEARD 213 00:11:07,994 --> 00:11:11,397 OF GASDERMIN-B, NO KNOWN 214 00:11:11,397 --> 00:11:11,831 FUNCTION. 215 00:11:11,831 --> 00:11:15,668 AND SO THE QUESTION WE WERE 216 00:11:15,668 --> 00:11:24,644 FACED WITHS THAT GASDERMIN-D 217 00:11:24,644 --> 00:11:26,279 RESPOND FOR RELEASE OF 218 00:11:26,279 --> 00:11:27,380 LEADERLESS CYTOKINES, ADDRESSED 219 00:11:27,380 --> 00:11:29,816 WITH SIMPLE EXPERIMENT. 220 00:11:29,816 --> 00:11:32,218 REMEMBER, WE CAN MONITOR 221 00:11:32,218 --> 00:11:33,286 PROTEOLYTIC MATURATION OF IL-1 222 00:11:33,286 --> 00:11:36,923 BETA, SO IT GOES FROM THE PRO 223 00:11:36,923 --> 00:11:39,425 FORM, ABOUT 31KD, TO MATURE 224 00:11:39,425 --> 00:11:40,293 FORM, 17 KD. 225 00:11:40,293 --> 00:11:41,594 MATURE FORM DOESN'T HAVE A 226 00:11:41,594 --> 00:11:48,968 SIGNAL PEP TIDE BUT WE CAN 227 00:11:48,968 --> 00:11:49,936 MONITOR FROM PRO TO MATURE BY 228 00:11:49,936 --> 00:12:00,380 WESTERN BLOT AND MONITOR RELEASE 229 00:12:00,380 --> 00:12:01,547 BY ELISA. 230 00:12:01,547 --> 00:12:05,251 LET'S COMPARE. 231 00:12:05,251 --> 00:12:06,586 WILDTYPE CELLS, YOU'LL NOTICE 232 00:12:06,586 --> 00:12:09,856 THAT THERE'S A TRANSITION FROM 233 00:12:09,856 --> 00:12:12,925 THE 31K TO THE MATURE 17K FORM 234 00:12:12,925 --> 00:12:14,694 ON INFLAMMASOME ACTIVATION THAT 235 00:12:14,694 --> 00:12:19,932 IS SHOWN HERE. 236 00:12:19,932 --> 00:12:20,800 YOU'RE GETTING THE MATURE 17K 237 00:12:20,800 --> 00:12:22,769 FORM THAT. 238 00:12:22,769 --> 00:12:25,705 IS RELEASED INTO THE 239 00:12:25,705 --> 00:12:26,005 SUPERNATANT. 240 00:12:26,005 --> 00:12:29,342 SO IT'S BEHAVING AS EXPECTED. 241 00:12:29,342 --> 00:12:35,248 WE'RE GOING TO DO THE SAME 242 00:12:35,248 --> 00:12:37,984 EXPERIMENT IN GASDERMIN-D NULL 243 00:12:37,984 --> 00:12:38,418 MACROPHAGE. 244 00:12:38,418 --> 00:12:40,420 YOU GENERATE MATURE IL-1 BETA, B 245 00:12:40,420 --> 00:12:45,224 17, AS MUCH AS IN THE WILDTYPE. 246 00:12:45,224 --> 00:12:48,294 BUT WHEN YOU MEASURE BY ELISA, 247 00:12:48,294 --> 00:12:50,563 RELEASE IS GREATLY COMPROMISED. 248 00:12:50,563 --> 00:12:53,966 FROM THIS WE SURMISED THAT 249 00:12:53,966 --> 00:12:56,936 GASDERMIN-D, WHATEVER IT DOES, 250 00:12:56,936 --> 00:13:02,875 SOMEHOW MEDIATES RELEASE OF 251 00:13:02,875 --> 00:13:06,612 LEADERLESS CYTOKINES LIKE IL-1. 252 00:13:06,612 --> 00:13:11,317 THE OTHER UNEXPECTED FINDING WAS 253 00:13:11,317 --> 00:13:12,485 GASDERMIN-D UNDERWENT CLEAVAGE 254 00:13:12,485 --> 00:13:16,122 ON INFLAMMASOME ACTIVATION. 255 00:13:16,122 --> 00:13:20,393 I'LL SHOW YOU DATA FOR 256 00:13:20,393 --> 00:13:23,996 CASPASE-11, WESTERN BLOT, NULL 257 00:13:23,996 --> 00:13:24,664 MACROPHAGES. 258 00:13:24,664 --> 00:13:27,600 WESTERN BLOT FOR GASDERMIN-D, 259 00:13:27,600 --> 00:13:28,968 THE NULL STRAINS LACK THE BAND. 260 00:13:28,968 --> 00:13:32,438 IF YOU LOOK AT THE RIGHT BAND, 261 00:13:32,438 --> 00:13:33,873 IN THE WILDTYPE SITUATION, 262 00:13:33,873 --> 00:13:37,510 NOTICE WHAT HAPPENS TO WILDTYPE 263 00:13:37,510 --> 00:13:40,580 GASDERMIN-D WHEN WE STIMULATE 264 00:13:40,580 --> 00:13:42,115 THE INFLAMMASOME WITH LPS. 265 00:13:42,115 --> 00:13:45,952 THERE'S A DIMINUTION OF THE 266 00:13:45,952 --> 00:13:48,254 FILAMENT BAND AND CONCOMITANT 267 00:13:48,254 --> 00:13:50,556 APPEARANCE OF A P30 FRAGMENT. 268 00:13:50,556 --> 00:13:51,324 ANTIBODY SPECIFIC FOR P30, 269 00:13:51,324 --> 00:13:53,626 THAT'S WHAT WE SEE. 270 00:13:53,626 --> 00:13:55,928 THAT DOESN'T HAPPEN WHEN 271 00:13:55,928 --> 00:13:59,098 CASPASE-11 NULL MACROPHAGES ARE 272 00:13:59,098 --> 00:14:02,068 STIMULATED WITH LPS. 273 00:14:02,068 --> 00:14:09,175 SO, FROM THIS, WE SURMISE THAT 274 00:14:09,175 --> 00:14:10,376 CASPASE-11 ON ACTIVATION LIKELY 275 00:14:10,376 --> 00:14:13,112 CLEAVES GASDERMIN-D TO GIVE YOU 276 00:14:13,112 --> 00:14:14,213 THIS P30 FRAGMENT. 277 00:14:14,213 --> 00:14:15,748 THE ANTIBODY WAS SPECIFIC FOR 278 00:14:15,748 --> 00:14:17,817 P30 SO WE DIDN'T SEE THE OTHER 279 00:14:17,817 --> 00:14:19,152 P20 FRAGMENT. 280 00:14:19,152 --> 00:14:21,654 BUT IF YOU TAKE RECOMBINANT 281 00:14:21,654 --> 00:14:26,893 GASDERMIN-D AND CASPASE-11, YOU 282 00:14:26,893 --> 00:14:27,760 GET EMERGENCE OF FRAGMENT. 283 00:14:27,760 --> 00:14:35,201 IF YOU MAP THE CLEAVAGE SITE, 284 00:14:35,201 --> 00:14:36,736 CLEAVAGE IS RESIDUE, 276. 285 00:14:36,736 --> 00:14:43,409 SO WHAT THIS DATA SAYS IS THAT 286 00:14:43,409 --> 00:14:43,976 ON INFLAMMASOME ACTIVATION, 287 00:14:43,976 --> 00:14:46,379 GASDERMIN-D HAS BEEN CLEAVED TO 288 00:14:46,379 --> 00:14:47,813 P30 AND P20. 289 00:14:47,813 --> 00:14:50,650 IT DOESN'T SAY WHETHER THAT 290 00:14:50,650 --> 00:14:52,418 CLEAVAGE IS NECESSARY FOR 291 00:14:52,418 --> 00:14:53,252 RELEASE OF IL-1 BETA. 292 00:14:53,252 --> 00:14:55,688 FOR THAT WE HAVE TO UNDERTAKE 293 00:14:55,688 --> 00:14:59,392 THE NEXT EXPERIMENT IN WHICH WE 294 00:14:59,392 --> 00:15:02,361 GENERATE A NON-CLEAVABLE FORM OF 295 00:15:02,361 --> 00:15:09,035 GASDERMIN-D, WHERE WE MAKE A 296 00:15:09,035 --> 00:15:13,406 KNOCK-IN MOUSE, WHERE IT IS 297 00:15:13,406 --> 00:15:16,142 CONVERTED TO ALLENASE, IS 298 00:15:16,142 --> 00:15:16,776 GASDERMIN-D NECESSARY AND 299 00:15:16,776 --> 00:15:21,380 CLEAVAGE NECESSARY FOR RELEASE 300 00:15:21,380 --> 00:15:22,815 OF LEADERLESS CYTOKINES? 301 00:15:22,815 --> 00:15:25,484 SO WE ISOLATE MACROPHAGES FROM 302 00:15:25,484 --> 00:15:27,320 WILDTYPE CELLS AND STIMULATE 303 00:15:27,320 --> 00:15:29,622 WITH LPS AND THEN MAKE -- 304 00:15:29,622 --> 00:15:32,491 RELEASE A TON OF IL-1 BETA. 305 00:15:32,491 --> 00:15:33,459 THE GASDERMIN-D, THE KNOCKOUT, 306 00:15:33,459 --> 00:15:35,561 AS I SHOWED BEFORE, THEY DON'T 307 00:15:35,561 --> 00:15:38,297 RELEASE IL-1 BETA. 308 00:15:38,297 --> 00:15:40,032 BUT LOOK AT THIS. 309 00:15:40,032 --> 00:15:42,435 THE MICE EXPRESSING THE 310 00:15:42,435 --> 00:15:43,836 PROCESSING MUTANT, SO EXPRESSING 311 00:15:43,836 --> 00:15:48,007 GASDERMIN-D, BUT WHICH CANNOT BE 312 00:15:48,007 --> 00:15:49,976 CLEAVED, ALSO CANNOT RELEASE 313 00:15:49,976 --> 00:15:50,977 IL-1 BETA. 314 00:15:50,977 --> 00:15:54,146 SO, WHAT THIS SAYS IS THAT NOT 315 00:15:54,146 --> 00:15:57,083 ONLY IS GASDERMIN-D REQUIRED FOR 316 00:15:57,083 --> 00:16:00,152 THE RELEASE OF LEADERLESS 317 00:16:00,152 --> 00:16:01,354 CYTOKINES LIKE IL-1 BETA AND 318 00:16:01,354 --> 00:16:05,291 IL-1, 18. 319 00:16:05,291 --> 00:16:11,831 IT HAS COULD BE CLEAVED, OR YOU 320 00:16:11,831 --> 00:16:13,266 DON'T GET CLEAVED. 321 00:16:13,266 --> 00:16:16,869 IT DAWNED ON US THEN MAYBE WE'RE 322 00:16:16,869 --> 00:16:19,472 DEALING WITH CHANNEL FORMING 323 00:16:19,472 --> 00:16:20,039 PROTEIN HERE. 324 00:16:20,039 --> 00:16:23,542 AND WE TESTED THAT AGAINST QUITE 325 00:16:23,542 --> 00:16:26,178 SIMPLY THAT WE SAID MAYBE ONE OF 326 00:16:26,178 --> 00:16:29,682 THE FRAGMENTS, P30 OR P20, IS 327 00:16:29,682 --> 00:16:30,750 ASSEMBLING INTO A PERFORATOR 328 00:16:30,750 --> 00:16:33,753 THAT ALLOWS FOR THE RELEASE OF 329 00:16:33,753 --> 00:16:34,420 IL-1 BETA. 330 00:16:34,420 --> 00:16:37,023 AND IN ORDER TO ADDRESS THAT 331 00:16:37,023 --> 00:16:39,458 QUESTION, WE JUST OVEREXPRESSED 332 00:16:39,458 --> 00:16:42,995 THE FULL LENGTH GASDERMIN-D P30 333 00:16:42,995 --> 00:16:46,565 OR P20 AND EXAMINED THE UPTAKE 334 00:16:46,565 --> 00:16:48,534 OF NUCLEAR DYE. 335 00:16:48,534 --> 00:16:51,470 WHEN YOU OVEREXPRESS P30, THE 336 00:16:51,470 --> 00:16:52,905 NUCLEAR DYE IS UPTAKEN, 337 00:16:52,905 --> 00:16:57,076 SUGGESTING THAT IT IS P30 THAT 338 00:16:57,076 --> 00:16:59,712 IS RESPONSIBLE FOR MEMBRANE 339 00:16:59,712 --> 00:17:01,580 DISRUPTION. 340 00:17:01,580 --> 00:17:03,883 SO TO SUMMARIZE, CASPASE-11 341 00:17:03,883 --> 00:17:05,651 CLEAVES GASDERMIN-D, YOU 342 00:17:05,651 --> 00:17:07,586 GENERATE THE P30 FRAGMENT, AND 343 00:17:07,586 --> 00:17:14,694 THIS IS THE P30 FRAGMENT, THE 344 00:17:14,694 --> 00:17:15,027 PERFORATOR. 345 00:17:15,027 --> 00:17:15,494 OKAY. 346 00:17:15,494 --> 00:17:22,935 SO, WE WERE QUITE INTERESTED IN 347 00:17:22,935 --> 00:17:25,404 THE MECHANISM THAT ASSEMBLED THE 348 00:17:25,404 --> 00:17:30,776 CHANNEL, P30, THE EVIDENCE WAS 349 00:17:30,776 --> 00:17:31,043 POSITIVE. 350 00:17:31,043 --> 00:17:33,212 IF WE TOOK PURIFIED LIPOSOMES, 351 00:17:33,212 --> 00:17:36,248 INCUBATED THEM WITH THE P30 352 00:17:36,248 --> 00:17:38,551 FRAGMENT OF GASDERMIN-D, AND 353 00:17:38,551 --> 00:17:41,287 EXAMINED THE PREPARATION BY 354 00:17:41,287 --> 00:17:42,521 NEGATIVE STAIN ELECTRON 355 00:17:42,521 --> 00:17:43,456 MICROSCOPY, THE IMAGE WAS 356 00:17:43,456 --> 00:17:43,723 STUNNING. 357 00:17:43,723 --> 00:17:49,195 WE COULD SEE THE LIPOSOME 358 00:17:49,195 --> 00:17:51,764 MEMBRANES, WITH WITHIN IT COULD 359 00:17:51,764 --> 00:17:55,601 SEE ASSEMBLED PORE-LIKE 360 00:17:55,601 --> 00:17:56,802 STRUCTURED, CALCULATED IT WAS 24 361 00:17:56,802 --> 00:17:57,570 P30s COMING TOGETHER AND 362 00:17:57,570 --> 00:18:01,474 GIVING YOU A PORE OF ABOUT 15 TO 363 00:18:01,474 --> 00:18:04,443 20 NANOMETERS INTERNAL DIAMETER, 364 00:18:04,443 --> 00:18:05,745 SO FAIRLY LARGE PORE. 365 00:18:05,745 --> 00:18:07,413 NOW, BY THIS TIME THE CAT WAS 366 00:18:07,413 --> 00:18:09,048 OUT OF THE BAG, AND THERE WAS A 367 00:18:09,048 --> 00:18:13,586 LOT OF COMP -- COMPETITION ON 368 00:18:13,586 --> 00:18:15,488 THE HORIZON. 369 00:18:15,488 --> 00:18:16,756 THE ATOMIC STRUCTURE WAS 370 00:18:16,756 --> 00:18:18,491 GENERATED BY WU AND COLLEAGUES 371 00:18:18,491 --> 00:18:24,530 AT CAR HARD, BUT ESSENTIALLY 372 00:18:24,530 --> 00:18:25,398 IT -- HARVARD. 373 00:18:25,398 --> 00:18:27,933 THE FULL LENGTH MOLECULE IS P30 374 00:18:27,933 --> 00:18:30,870 AND P20, AND P30 IS HELD IN 375 00:18:30,870 --> 00:18:32,204 CONSTRAINED MANNER BY P20. 376 00:18:32,204 --> 00:18:35,241 ONCE YOU HAVE THIS PROTEOLYTIC 377 00:18:35,241 --> 00:18:40,079 CLEAVAGE, P20 FLIES OFF, AND P30 378 00:18:40,079 --> 00:18:40,613 UNDERGOES ENORMOUS 379 00:18:40,613 --> 00:18:41,680 CONFIRMATIONAL CHANGE. 380 00:18:41,680 --> 00:18:46,318 THERE'S SORT OF AN UNVEILING, 381 00:18:46,318 --> 00:18:48,320 UNFURLING OF THIS N-TERMINUS 382 00:18:48,320 --> 00:18:50,289 INTO BETA SHEETS THAT'S INSERTED 383 00:18:50,289 --> 00:18:51,924 INTO THE MEMBRANE. 384 00:18:51,924 --> 00:19:00,900 THIS IS HOW WU'S STRUCTURE, 33 385 00:19:00,900 --> 00:19:01,834 P30S COMING TOGETHER. 386 00:19:01,834 --> 00:19:03,769 I THINK THE PROBLEM WAS SOLVED 387 00:19:03,769 --> 00:19:08,541 IN A SENSE THAT THE PORE IS 388 00:19:08,541 --> 00:19:09,675 GENERATED, THE CHANNEL IS 389 00:19:09,675 --> 00:19:12,845 GENERATED BY THIS WHAT WAS AN 390 00:19:12,845 --> 00:19:15,581 UNKNOWN PROTEIN IN 2015, 391 00:19:15,581 --> 00:19:17,183 GASDERMIN-D, GENERATED BY P30 392 00:19:17,183 --> 00:19:20,419 FRAGMENT COMING TOGETHER, 393 00:19:20,419 --> 00:19:22,354 OLIGOMERRIZING AND GIVING YOU A 394 00:19:22,354 --> 00:19:24,089 PORE IN THE MEMBRANE. 395 00:19:24,089 --> 00:19:25,624 AND SO THE ANSWER TO THE 396 00:19:25,624 --> 00:19:28,360 QUESTION OF HOW A LEADERLESS 397 00:19:28,360 --> 00:19:30,062 CYTOKINE LIKE IL-1 BETA AND 398 00:19:30,062 --> 00:19:35,267 IL-18 RELEASE I THINK IS NOW 399 00:19:35,267 --> 00:19:38,471 ANSWERED, WHETHER YOU ACTIVATE 400 00:19:38,471 --> 00:19:43,275 OR CANONICAL OR NON-CANONICAL 401 00:19:43,275 --> 00:19:45,478 PATHWAY, AS LONG AS YOU HAVE 402 00:19:45,478 --> 00:19:52,017 CASPASES, THIS WILL LEAD TO 403 00:19:52,017 --> 00:19:56,856 CLEAVAGE OF GASDERMIN-D, FORMING 404 00:19:56,856 --> 00:19:58,491 A PORE, LARGE ENOUGH THAT IT'S 405 00:19:58,491 --> 00:20:01,760 GOING TO ALLOW FOR PASSAGE OF 406 00:20:01,760 --> 00:20:04,263 4.5-NANOMETER IL-1 BETA AND 407 00:20:04,263 --> 00:20:04,463 IL-18. 408 00:20:04,463 --> 00:20:07,132 OBVIOUSLY IF THE PORE IS 409 00:20:07,132 --> 00:20:09,401 PERSISTENT, YOU'LL COLLAPSE THE 410 00:20:09,401 --> 00:20:10,135 ELECTRO CHEMICAL GRADIENT, 411 00:20:10,135 --> 00:20:14,139 LEADING TO LYSIS OF THE CELLS 412 00:20:14,139 --> 00:20:17,409 WHICH ARE GENERALLY MEASURED BY 413 00:20:17,409 --> 00:20:26,819 LDH RELEASE. 414 00:20:26,819 --> 00:20:28,254 TWO PAPERS ANNOUNCING DISCOVERY 415 00:20:28,254 --> 00:20:31,524 FROM MY OWN GROUP AND A GROUP 416 00:20:31,524 --> 00:20:36,562 USING DIFFERENT METHODS, FORWARD 417 00:20:36,562 --> 00:20:38,964 GENETICS, HE USE CRISPR, SAME 418 00:20:38,964 --> 00:20:41,834 CONCLUSIONS, PUBLISHED IN 2015, 419 00:20:41,834 --> 00:20:43,569 SINCE THEN STUDY OF GASDERMINS 420 00:20:43,569 --> 00:20:47,273 HAVE BEEN A COTTAGE INDUSTRY 421 00:20:47,273 --> 00:20:48,707 WITH A FLURRY, EXPLOSION OF 422 00:20:48,707 --> 00:20:49,708 PUBLICATION. 423 00:20:49,708 --> 00:20:51,944 I WANTED TO BRING YOUR ATTENTION 424 00:20:51,944 --> 00:20:54,413 TO ONE PUBLICATION FROM A COUPLE 425 00:20:54,413 --> 00:20:56,916 YEARS AGO, WHICH WAS THE 426 00:20:56,916 --> 00:20:58,250 DISCOVERY THAT BACTERIA HAVE 427 00:20:58,250 --> 00:21:02,054 GASDERMIN. 428 00:21:02,054 --> 00:21:04,590 AND QUITE REMARKABLY, THE 429 00:21:04,590 --> 00:21:06,559 BACTERIAL GASDERMIN EXISTS ON AN 430 00:21:06,559 --> 00:21:10,763 OPERON IN WHICH IS ALSO ENCODED 431 00:21:10,763 --> 00:21:12,932 A CASPASE-LIKE PROTEASE, SO ON 432 00:21:12,932 --> 00:21:17,536 PHAGE INFECTION THE CASPASE-LIKE 433 00:21:17,536 --> 00:21:19,271 PROTEASES IS ACTIVATED, 434 00:21:19,271 --> 00:21:21,373 BACTERIAL GASDERMIN IS CLEAVED, 435 00:21:21,373 --> 00:21:22,575 N-TERMINAL DOMAIN OF BACTERIAL 436 00:21:22,575 --> 00:21:23,876 GASDERMIN MAKES A PORE IN THE 437 00:21:23,876 --> 00:21:25,311 MEMBRANE OF THE BACTERIA, 438 00:21:25,311 --> 00:21:35,387 LEADING TO ITS DEMISE, AND THE 439 00:21:35,387 --> 00:21:36,889 REPLICATIVE NICHE IS DENIED. 440 00:21:36,889 --> 00:21:38,457 SO, THIS I FIND IS QUITE 441 00:21:38,457 --> 00:21:40,826 REMARKABLE BECAUSE WHEN WE THINK 442 00:21:40,826 --> 00:21:41,594 OF BACTERIAL INNATE IMMUNITY, 443 00:21:41,594 --> 00:21:44,229 YOU KNOW, IT'S BEEN VERY 444 00:21:44,229 --> 00:21:47,933 POWERFUL IN TERMS OF TOOLS IT 445 00:21:47,933 --> 00:21:51,236 HAS GENERATED. 446 00:21:51,236 --> 00:21:53,973 RESTRICTION ENZYMES, CRISPR. 447 00:21:53,973 --> 00:21:56,275 BUT YOU AND I, THEY ARE NOT 448 00:21:56,275 --> 00:21:58,210 CONSERVED ENOUGH, WE DON'T HAVE 449 00:21:58,210 --> 00:21:59,311 RESTRICTION ENZYMES OR CRISPR, 450 00:21:59,311 --> 00:22:03,148 BUT HERE IS A MECHANISM OF 451 00:22:03,148 --> 00:22:04,049 INNATE IMMUNITY IN BACTERIA 452 00:22:04,049 --> 00:22:10,322 THAT'S PRESENT IN YOU AND I. 453 00:22:10,322 --> 00:22:11,924 QUITE REMARKABLE. 454 00:22:11,924 --> 00:22:14,893 ALL RIGHT. 455 00:22:14,893 --> 00:22:18,297 SO, NOW, OBVIOUSLY FROM WHAT 456 00:22:18,297 --> 00:22:22,768 I'VE INTONATED THROUGH THIS 457 00:22:22,768 --> 00:22:24,637 GASDERMIN-D PORE COMES OUT A 458 00:22:24,637 --> 00:22:25,738 PLETHORA OF CYTOKINES. 459 00:22:25,738 --> 00:22:27,506 ONCE YOU FORM THIS NOT ONLY DO 460 00:22:27,506 --> 00:22:31,543 YOU GET IL-1 BETA AND IL-18, 461 00:22:31,543 --> 00:22:32,845 IL-1 ALPHA, OTHER 462 00:22:32,845 --> 00:22:33,846 PRO-INFLAMMATORY SUBSTANCES THAT 463 00:22:33,846 --> 00:22:38,884 POUR OUT, AND THIS IS REALLY 464 00:22:38,884 --> 00:22:39,652 CONTRIBUTES TO CYTOKINE STORM 465 00:22:39,652 --> 00:22:43,355 RESPONSIBLE FOR SO MANY CRITICAL 466 00:22:43,355 --> 00:22:46,225 ILLNESSES LIKE SEPSIS AND ARDS. 467 00:22:46,225 --> 00:22:47,626 AND I THINK THE NOTION IN OUR 468 00:22:47,626 --> 00:22:50,829 MIND IS THAT IF ONE COULD 469 00:22:50,829 --> 00:22:54,366 DEVELOP AN INHIBITOR TO 470 00:22:54,366 --> 00:22:57,503 GASDERMIN-D, IN ONE FELL SWOOP 471 00:22:57,503 --> 00:22:59,238 YOU WOULD ATTENUATE THE RELEASE 472 00:22:59,238 --> 00:23:06,912 OF THESE CYTOKINES, IT MAY BE A 473 00:23:06,912 --> 00:23:12,651 BENEFIT IN THESE ACUTE 474 00:23:12,651 --> 00:23:12,918 SYNDROMES. 475 00:23:12,918 --> 00:23:15,688 THERE'S A LOT OF THE ACTIVITY 476 00:23:15,688 --> 00:23:20,826 TRYING TO DEVELOP 477 00:23:20,826 --> 00:23:21,226 PHARMACOLOGICALLY, 478 00:23:21,226 --> 00:23:28,200 THERAPEUTICALLY EFFICACIOUS, 479 00:23:28,200 --> 00:23:28,967 INHIBITORS THAT HAVE 480 00:23:28,967 --> 00:23:29,268 SPECIFICITY. 481 00:23:29,268 --> 00:23:32,371 NO I'M GOING TO SOMETHING 482 00:23:32,371 --> 00:23:32,671 UNEXPECTED. 483 00:23:32,671 --> 00:23:35,107 I'LL STILL NOT SURE OF THE 484 00:23:35,107 --> 00:23:37,309 RAMIFICATIONS OF THIS DISCOVERY 485 00:23:37,309 --> 00:23:39,278 SO I THOUGHT I WOULD SHARE WITH 486 00:23:39,278 --> 00:23:39,511 YOU. 487 00:23:39,511 --> 00:23:40,913 WHEN WE DO SCREENS OF TENS OF 488 00:23:40,913 --> 00:23:42,014 THOUSANDS OF MICE, REMEMBER WHAT 489 00:23:42,014 --> 00:23:45,751 WE'RE DOING IS WE'RE GIVING THEM 490 00:23:45,751 --> 00:23:47,453 INDUSTRIAL LOADS OF CYTOSOLIC 491 00:23:47,453 --> 00:23:49,788 LPS, SO WE CAN MEASURE THE 492 00:23:49,788 --> 00:23:51,557 RELEASE OF IL-1 BETA, THEY MAKE 493 00:23:51,557 --> 00:23:53,192 BUCKET LOADS OF IL-1 BETA, AND 494 00:23:53,192 --> 00:23:55,060 WE'RE LOOKING FOR SITUATIONS 495 00:23:55,060 --> 00:23:58,530 WHERE THE INFLAMMASOME IS 496 00:23:58,530 --> 00:23:58,764 BROKEN. 497 00:23:58,764 --> 00:24:00,632 THAT'S THE ASSAY. 498 00:24:00,632 --> 00:24:02,935 BUT, THESE CELLS IN WHICH WE'VE 499 00:24:02,935 --> 00:24:05,003 GIVEN -- EXPOSED TO INDUSTRIAL 500 00:24:05,003 --> 00:24:06,872 LEVELS OF CYTOSOLIC LPS ARE 501 00:24:06,872 --> 00:24:08,607 DESTINED TO DIE. 502 00:24:08,607 --> 00:24:11,777 HAVE YOU PRECIPITOUS ACTIVATION 503 00:24:11,777 --> 00:24:14,079 OF INFLAMMATORY CASPASES. 504 00:24:14,079 --> 00:24:16,048 GENERATION OF GASDERMIN-D PORES. 505 00:24:16,048 --> 00:24:19,218 AND THEN THE CELLS LYSE. 506 00:24:19,218 --> 00:24:22,855 A SANITY CHECK, WE MEASURE LDH 507 00:24:22,855 --> 00:24:23,188 RELEASE. 508 00:24:23,188 --> 00:24:26,125 EVERY TIME YOU GET INDUSTRIAL 509 00:24:26,125 --> 00:24:28,193 AMOUNTS OF IL-1 BETA RELEASE YOU 510 00:24:28,193 --> 00:24:29,528 GET HDA RELEASE. 511 00:24:29,528 --> 00:24:32,931 OKAY, WELL, YEAH, THAT'S A 512 00:24:32,931 --> 00:24:34,466 SANITY CHECK. 513 00:24:34,466 --> 00:24:36,101 IT CAME TO ME ONE THEY, THEY 514 00:24:36,101 --> 00:24:39,071 SAID WE'VE GOT A STRAIN THAT 515 00:24:39,071 --> 00:24:41,940 RELEASES IL-1 BETA. 516 00:24:41,940 --> 00:24:45,410 BUT DOESN'T RELEASE LDH. 517 00:24:45,410 --> 00:24:48,380 IT'S A STRAIN, MUTATION, THIS 518 00:24:48,380 --> 00:24:50,549 NINJ1 PROTEIN. 519 00:24:50,549 --> 00:24:52,417 HOW CAN THAT BE? 520 00:24:52,417 --> 00:24:54,386 ARE THE CELLS DEAD? 521 00:24:54,386 --> 00:24:55,254 YEAH, THEY ARE DEAD. 522 00:24:55,254 --> 00:25:03,462 I SAID, YOU'RE TELLING ME THAT 523 00:25:03,462 --> 00:25:05,097 YOU'RE MEASURING CELLS FROM LDH, 524 00:25:05,097 --> 00:25:06,999 YOU DON'T SEE A SIGNAL? 525 00:25:06,999 --> 00:25:09,401 LDH IS USED IN THE SIGNAL, THE 526 00:25:09,401 --> 00:25:14,306 CLINIC, IT'S HOW WE MEASURE CELL 527 00:25:14,306 --> 00:25:15,274 LYSIS. 528 00:25:15,274 --> 00:25:16,842 DEAD CELLS LYSE, RELEASE LDH. 529 00:25:16,842 --> 00:25:17,910 YOU'RE NOT TO TELL ME YOU HAVE 530 00:25:17,910 --> 00:25:21,413 DEAD CELLS THAT ARE NOT 531 00:25:21,413 --> 00:25:22,181 RELEASING LDH. 532 00:25:22,181 --> 00:25:25,450 JUST CAN'T BE. 533 00:25:25,450 --> 00:25:25,918 RIGHT? 534 00:25:25,918 --> 00:25:29,955 DOESN'T MAKE ANY SENSE. 535 00:25:29,955 --> 00:25:33,992 SO, ONE THING I SHOULD SAY IL-1 536 00:25:33,992 --> 00:25:38,797 BETA IS RELATIVELY SMALL, 17KD, 537 00:25:38,797 --> 00:25:41,767 LDH IS BIG, 140KD. 538 00:25:41,767 --> 00:25:44,603 NOW, WE'VE GOTTEN NINJ1 FROM A 539 00:25:44,603 --> 00:25:45,804 FORWARD GENETIC SCREENING, IT'S 540 00:25:45,804 --> 00:25:47,673 NOT GETTING INTO THE GENETICS, 541 00:25:47,673 --> 00:25:51,176 OFTEN THE STRAINS CARRY MULTIPLE 542 00:25:51,176 --> 00:25:51,577 MUTATIONS. 543 00:25:51,577 --> 00:25:54,680 I'M NOT GOING TO BELIEVE THIS 544 00:25:54,680 --> 00:25:56,849 TILL YOU MAKE A KNOCKOUT. 545 00:25:56,849 --> 00:26:02,020 MAYBE TWO OR THREE INDEPENDENT 546 00:26:02,020 --> 00:26:04,289 KNOCKOUT STRAINS TO BE SURE 547 00:26:04,289 --> 00:26:06,391 BECAUSE THIS IS WHACK-O. 548 00:26:06,391 --> 00:26:07,793 HERE IS THE DATA. 549 00:26:07,793 --> 00:26:10,429 SO, WE'RE GOING TO LOOK AT IL-1 550 00:26:10,429 --> 00:26:13,599 BETA RELEASE, OPEN BARS ARE 551 00:26:13,599 --> 00:26:16,034 WILDTYPE MACROPHAGES, RED BARS 552 00:26:16,034 --> 00:26:21,340 ARE NINJ1 NULL MACROPHAGES, 553 00:26:21,340 --> 00:26:25,777 STIMULATE WITH LPS, IN WILD TYPE 554 00:26:25,777 --> 00:26:36,321 AND NINJ1 KNOCKOUT YOU'RE IL-1 555 00:26:38,023 --> 00:26:39,358 BETA RELEASE. 556 00:26:39,358 --> 00:26:41,894 WE SEE THAT THE GENERATION OF 557 00:26:41,894 --> 00:26:45,731 THE P30 FRAGMENT IN WILD TYPE 558 00:26:45,731 --> 00:26:47,699 AND NINJ1 NULL MACROPHAGES OF 559 00:26:47,699 --> 00:26:50,135 GASDERMIN-D IS EQUIVALENT. 560 00:26:50,135 --> 00:26:56,475 IN THE NINJ1 NULL MACROPHAGES 561 00:26:56,475 --> 00:26:57,809 THAT ARE DYING YOU'RE GETTING 562 00:26:57,809 --> 00:27:00,178 GASDERMIN-D PORES. 563 00:27:00,178 --> 00:27:05,017 LET'S LOOK AT LDH RELEASE. 564 00:27:05,017 --> 00:27:08,654 WILD TYPE MACROPHAGE RELEASED, 565 00:27:08,654 --> 00:27:10,255 NULL MACROPHAGES DO NOT. 566 00:27:10,255 --> 00:27:14,326 I'M LIKE, MAN, THIS IS JUST 567 00:27:14,326 --> 00:27:16,295 SOMETHING WEIRD ABOUT LDH. 568 00:27:16,295 --> 00:27:21,233 I JUST CANNOT BUY THIS RESULT. 569 00:27:21,233 --> 00:27:23,335 BUT THEN WE LOOKED AT THE 570 00:27:23,335 --> 00:27:24,303 SECRETOME. 571 00:27:24,303 --> 00:27:28,273 WE LOOKED AT THE RELEASE OF 572 00:27:28,273 --> 00:27:32,411 UNBIASED, REALLY OF ALL PROTEINS 573 00:27:32,411 --> 00:27:33,845 RELEASED INTO THE EXTRACELLULAR 574 00:27:33,845 --> 00:27:35,948 MEDIA BY LPS. 575 00:27:35,948 --> 00:27:38,884 INDEED, IN THE ABSENCE OF NINJ1, 576 00:27:38,884 --> 00:27:40,852 THERE'S A LARGE SUBSTANTIAL 577 00:27:40,852 --> 00:27:43,055 NUMBER OF PROTEINS WHOSE RELEASE 578 00:27:43,055 --> 00:27:45,324 IS COMPROMISED. 579 00:27:45,324 --> 00:27:53,465 SO IT'S NOT JUST LDH. 580 00:27:53,465 --> 00:27:54,566 I'M LIKE, YOU KNOW, THIS IS 581 00:27:54,566 --> 00:27:57,202 REALLY WEIRD. 582 00:27:57,202 --> 00:28:01,573 WE'VE GOT TO LOOK AT THE CELLS. 583 00:28:01,573 --> 00:28:06,411 SO, I'LL JUST REMIND YOU THAT 584 00:28:06,411 --> 00:28:07,813 CELLS WERE GIVEN NECROTIC 585 00:28:07,813 --> 00:28:17,055 STIMULUS, WE'RE LOOKING AT BONE 586 00:28:17,055 --> 00:28:19,491 MARROW DERIVED MACROPHAGES, 587 00:28:19,491 --> 00:28:21,126 INITIALLY BALLOON AND COLLAPSE. 588 00:28:21,126 --> 00:28:22,527 LET'S ORIENT OURSELVES WITH THAT 589 00:28:22,527 --> 00:28:22,861 IMAGE. 590 00:28:22,861 --> 00:28:23,729 THEY BALLOON UP. 591 00:28:23,729 --> 00:28:27,766 AND THEN THEY COLLAPSE. 592 00:28:27,766 --> 00:28:28,767 THEY UNDERGO LYSIS. 593 00:28:28,767 --> 00:28:33,105 NOW WE'RE GOING TO LOOK AT A LOW 594 00:28:33,105 --> 00:28:39,378 POWER VIEW OF BONE MARROW 595 00:28:39,378 --> 00:28:40,445 DERIVED MACROPHAGES, NIGERICIN. 596 00:28:40,445 --> 00:28:45,717 THEY BALLOON BUT COLLAPSE INTO 597 00:28:45,717 --> 00:28:46,618 THE SCRAMBLED EGG APPEARANCE. 598 00:28:46,618 --> 00:28:49,254 IF YOU DO THE EXPERIMENT WITH 599 00:28:49,254 --> 00:28:52,424 THE NINJ1 NULL CELLS THEY 600 00:28:52,424 --> 00:28:58,130 BALLOON, BALLOON UP, AND BALLOON 601 00:28:58,130 --> 00:28:58,296 UP. 602 00:28:58,296 --> 00:28:59,431 THIS IS OVERNIGHT. 603 00:28:59,431 --> 00:29:02,934 IT'S NOT LIKE WE SAID, YOU KNOW, 604 00:29:02,934 --> 00:29:05,070 WE'VE GOT A WINDOW OF 10 MINUTES 605 00:29:05,070 --> 00:29:07,606 TO MAKE THIS FIGURE. 606 00:29:07,606 --> 00:29:10,509 NO, YOU CAN GO HOME, SPEND THE 607 00:29:10,509 --> 00:29:12,878 NIGHT, COME BACK, THEY ARE STILL 608 00:29:12,878 --> 00:29:13,145 BALLOONED. 609 00:29:13,145 --> 00:29:14,713 EVENTUALLY OF COURSE THEY WILL 610 00:29:14,713 --> 00:29:14,946 LYSE. 611 00:29:14,946 --> 00:29:19,551 BUT THE WINDOW OF BALLOONING IS 612 00:29:19,551 --> 00:29:19,951 ENORMOUS. 613 00:29:19,951 --> 00:29:22,287 I REMEMBER WHEN WE FIRST 614 00:29:22,287 --> 00:29:25,123 PUBLISHED THIS PAPER, BECAUSE OF 615 00:29:25,123 --> 00:29:27,459 TIME, CRISPR, PEOPLE DIDN'T 616 00:29:27,459 --> 00:29:29,795 BELIEVE IT. 617 00:29:29,795 --> 00:29:30,128 IT'S BULLSHIT. 618 00:29:30,128 --> 00:29:31,663 EVERYBODY DID A CRISPR 619 00:29:31,663 --> 00:29:33,565 EXPERIMENT, INDEED ABLE TO 620 00:29:33,565 --> 00:29:36,401 DUPLICATE THE WORK. 621 00:29:36,401 --> 00:29:39,237 NOW, THE BALLOONED CELLS ARE 622 00:29:39,237 --> 00:29:40,806 DEAD. 623 00:29:40,806 --> 00:29:42,774 THEY ARE DEADER THAN DOORNAILS. 624 00:29:42,774 --> 00:29:47,479 BUT THEY ARE BALLOONED. 625 00:29:47,479 --> 00:29:49,948 THIS IS NOT LYSING. 626 00:29:49,948 --> 00:29:51,316 VERY, VERY ODD. 627 00:29:51,316 --> 00:29:56,721 I'M LIKE, WHAT THE HELL IS 628 00:29:56,721 --> 00:30:01,326 NINJ1? 629 00:30:01,326 --> 00:30:04,162 AGAIN, UNEXPECTED DISCOVERY. 630 00:30:04,162 --> 00:30:11,169 LIBRARY TELLS YOU NOTHING. 631 00:30:11,169 --> 00:30:12,571 IT'S 157 AMINO ACIDS, SMALL 632 00:30:12,571 --> 00:30:12,971 PROTEIN. 633 00:30:12,971 --> 00:30:17,075 I'LL GET TO THIS LATER, ONE OF 634 00:30:17,075 --> 00:30:18,176 THE TOPOLOGY PROGRAMS PREDICTS 635 00:30:18,176 --> 00:30:23,215 IT TO HAVE THE N-TERMINUS AND 636 00:30:23,215 --> 00:30:27,252 C-TERMINUS OUT, AND WITH AMPHI 637 00:30:27,252 --> 00:30:31,089 PATTIC DOMAIN THAT'S EXPERIMENT. 638 00:30:31,089 --> 00:30:36,228 IT'S CONSERVED. 639 00:30:36,228 --> 00:30:38,296 DROSOPHILAS THAT THREE, NINJ A, 640 00:30:38,296 --> 00:30:41,466 B, C. 641 00:30:41,466 --> 00:30:43,235 WHAT IS IT DOING? 642 00:30:43,235 --> 00:30:51,643 SO, THIS GOT ME THINKING, US 643 00:30:51,643 --> 00:30:52,244 THINKING. 644 00:30:52,244 --> 00:30:54,613 MAYBE NINJ1 IS NOT ONLY 645 00:30:54,613 --> 00:30:56,915 DOWNSTREAM OF GASDERMIN-D FOR 646 00:30:56,915 --> 00:30:59,851 CELL LYSIS BUT IT'S THE FINAL 647 00:30:59,851 --> 00:31:06,858 COMMON COMPONENT THAT YOU NEED 648 00:31:06,858 --> 00:31:08,827 THIS FOR CELLS TO LYSE NO MATTER 649 00:31:08,827 --> 00:31:10,362 HOW THEY DIE. 650 00:31:10,362 --> 00:31:14,900 NO MATTER HOW THEY DIE YOU NEED 651 00:31:14,900 --> 00:31:16,201 NINJ1 FOR CELL LYSIS. 652 00:31:16,201 --> 00:31:18,069 PRETTY WILD. 653 00:31:18,069 --> 00:31:20,906 LET'S GO THROUGH PERCENTAGE LDH 654 00:31:20,906 --> 00:31:24,743 RELEASE, OPEN BARS ARE WILDTYPE 655 00:31:24,743 --> 00:31:27,913 CELLS, RED NINJ1 KNOCKOUTS. 656 00:31:27,913 --> 00:31:31,183 IN ABSENCE OF TREATMENT, THE 657 00:31:31,183 --> 00:31:41,459 RELEASE IS 18%. 658 00:31:43,161 --> 00:31:47,632 NIGERICIN, YOU SAW RELEASE OF 659 00:31:47,632 --> 00:31:53,438 NINJ1, NULL CELLS COMPROMISED, 660 00:31:53,438 --> 00:31:55,173 USED STREPTOLYSIN OR LISTERIA, 661 00:31:55,173 --> 00:32:00,679 BUT LOOK AT THIS, CYST CISPLATIN 662 00:32:00,679 --> 00:32:03,181 INDUCED DEATH HAS NOTHING TO DO 663 00:32:03,181 --> 00:32:04,382 WITH GASDERMIN-D, YET THOSE 664 00:32:04,382 --> 00:32:07,018 CELLS, THE LYSIS OF THOSE CELLS, 665 00:32:07,018 --> 00:32:13,358 THE LYSIS OF DEAD CELLS, FROM 666 00:32:13,358 --> 00:32:16,294 CISPLATIN EXPOSURE HAS A NINJ1 667 00:32:16,294 --> 00:32:18,597 DEPENDENCE. 668 00:32:18,597 --> 00:32:23,735 OKAY. 669 00:32:23,735 --> 00:32:28,340 SO, WHY IS THIS SO SURPRISING? 670 00:32:28,340 --> 00:32:33,912 BECAUSE YOU REMEMBER MISS JONES, 671 00:32:33,912 --> 00:32:35,814 OUR HIGH SCHOOL BIOLOGY TEACHER. 672 00:32:35,814 --> 00:32:39,517 SHE SAID WHEN CELLS DIE, THEY 673 00:32:39,517 --> 00:32:42,053 LYSE, AND IT'S A MECHANISM, A 674 00:32:42,053 --> 00:32:43,488 PASSIVE MECHANISM, AND WRITE IT 675 00:32:43,488 --> 00:32:45,857 DOWN IN YOUR NOTEBOOK, BOYS AND 676 00:32:45,857 --> 00:32:47,626 GIRLS, WE'LL CALL IT OSMOTIC 677 00:32:47,626 --> 00:32:49,294 LYSIS. 678 00:32:49,294 --> 00:32:50,695 THAT'S WHAT WE LEARNED. 679 00:32:50,695 --> 00:32:53,231 RIGHT? 680 00:32:53,231 --> 00:32:53,732 [LAUGHTER] 681 00:32:53,732 --> 00:32:55,934 THAT'S WHAT HAPPENS. 682 00:32:55,934 --> 00:33:01,406 YEAH. 683 00:33:01,406 --> 00:33:01,973 OKAY. 684 00:33:01,973 --> 00:33:08,413 WHAT I I'M SAYING IS SOMETHING 685 00:33:08,413 --> 00:33:10,048 RADICALLY DIFFERENT. 686 00:33:10,048 --> 00:33:12,017 LYSIS, EVEN FOLLOWING DEATH, IS 687 00:33:12,017 --> 00:33:18,490 AN ACTIVE EVENT AND IT'S A 688 00:33:18,490 --> 00:33:19,024 PROGRAMMED EVENT. 689 00:33:19,024 --> 00:33:23,194 NOW, HOW ARE WE GOING TO STUDY 690 00:33:23,194 --> 00:33:23,395 THIS? 691 00:33:23,395 --> 00:33:25,497 WELL, A PREDICTION WOULD BE IF 692 00:33:25,497 --> 00:33:26,831 NINJ1 REALLY IS RESPONSIBLE FOR 693 00:33:26,831 --> 00:33:28,867 CELL LYSIS, IF YOU OVEREXPRESS 694 00:33:28,867 --> 00:33:32,404 IT, IT SHOULD SHATTER CELLS. 695 00:33:32,404 --> 00:33:35,106 INDEED THAT'S THE CASE. 696 00:33:35,106 --> 00:33:41,046 TAKE 293T CELLS, A HUMAN NINJ1, 697 00:33:41,046 --> 00:33:44,316 FOR THAT MATTER FLY NINJ A OR B, 698 00:33:44,316 --> 00:33:47,919 YOU SHATTER THE CELLS. 699 00:33:47,919 --> 00:33:50,622 THIS BECOMES A SURROGATE 700 00:33:50,622 --> 00:33:52,190 BIOCHEMICAL ASSAY, BY WHICH WE 701 00:33:52,190 --> 00:33:54,259 CAN INTERROGATE IT THROUGH 702 00:33:54,259 --> 00:33:54,893 ALANINE SCANNING MUTAGENESIS 703 00:33:54,893 --> 00:33:57,329 I'LL SHOW YOU ON THE NEXT SLIDE. 704 00:33:57,329 --> 00:34:00,198 BUT ALSO I WANT TO EMPHASIZE 705 00:34:00,198 --> 00:34:02,801 THAT NINJ1 IN ITS VARIOUS GUISES 706 00:34:02,801 --> 00:34:05,670 IS PRESENT ALL THE WAY DOWN TO 707 00:34:05,670 --> 00:34:11,476 THE FLY. 708 00:34:11,476 --> 00:34:14,412 SO, AGAIN, NINJ1 GOING FROM THE 709 00:34:14,412 --> 00:34:18,116 N-TERMINUS TO C-TERMINUS HAS 710 00:34:18,116 --> 00:34:20,852 THIS EXTERNAL AMPIPATHIC HELIX, 711 00:34:20,852 --> 00:34:23,321 A TRANSMEMBRANE DOMAIN 1 AND 712 00:34:23,321 --> 00:34:26,324 TRANSMEMBRANE DOMAIN 2, IF YOU 713 00:34:26,324 --> 00:34:27,525 LOOK AT CONSERVATION SCORE, 714 00:34:27,525 --> 00:34:29,694 LOWER THE SCORE, HIGHER 715 00:34:29,694 --> 00:34:31,329 CONSERVATION, THE AMPIPATHIC 716 00:34:31,329 --> 00:34:33,098 HELIX IS HIGHLY CONSERVED AMONG 717 00:34:33,098 --> 00:34:33,331 SPECIES. 718 00:34:33,331 --> 00:34:36,801 NOW WE'RE GOING TO DO AN 719 00:34:36,801 --> 00:34:37,369 ALLOSCAN, CHANGE FIVE AMINO 720 00:34:37,369 --> 00:34:40,205 ACIDS AT A TIME TO ALANINE, 721 00:34:40,205 --> 00:34:41,639 STARTING FROM THE N-TERMINUS, 722 00:34:41,639 --> 00:34:43,174 GOING TO THE C-TERMINUS, AND 723 00:34:43,174 --> 00:34:44,609 WE'RE GOING TO ASSAY THE 724 00:34:44,609 --> 00:34:46,111 PROTEINS BY THEIR ABILITY TO 725 00:34:46,111 --> 00:34:47,779 INDUCE CELL DEATH. 726 00:34:47,779 --> 00:34:52,016 SO WHEN YOU OVEREXPRESS WILDTYPE 727 00:34:52,016 --> 00:34:55,453 NINJ1, YOU INDUCE CELL DEATH. 728 00:34:55,453 --> 00:35:00,525 IF YOU MUTATE THE N-TERMINUS, 729 00:35:00,525 --> 00:35:03,762 IT'S QUITE RESISTANT, QUITE 730 00:35:03,762 --> 00:35:04,529 ACCOMMODATING, TO MUTATION. 731 00:35:04,529 --> 00:35:08,166 BUT AS SOON AS YOU MESS WITH THE 732 00:35:08,166 --> 00:35:10,135 AMPIPATHIC HELIX, NINJ1 IS NO 733 00:35:10,135 --> 00:35:14,038 LONGER ABLE TO LYSE CELLS. 734 00:35:14,038 --> 00:35:17,976 SO THE AMPIPATHIC HEEL IS IS 735 00:35:17,976 --> 00:35:18,610 IMPORTANT. 736 00:35:18,610 --> 00:35:19,411 WHAT'S THE ENDOGENOUS MOLECULE 737 00:35:19,411 --> 00:35:21,613 LOOKING LIKE? 738 00:35:21,613 --> 00:35:23,014 HOW DOES THAT BEHAVE? 739 00:35:23,014 --> 00:35:26,084 SO, LET'S LOOK AT BONE 740 00:35:26,084 --> 00:35:27,185 MARROW-DERIVED MACROPHAGES. 741 00:35:27,185 --> 00:35:30,789 WE TREAT THEM WITH POISON 742 00:35:30,789 --> 00:35:33,191 NIGERICIN, ONE HOUR, TWO HOUR, 743 00:35:33,191 --> 00:35:35,160 BLOTTING FOR ENDOGENOUS NINJ1 744 00:35:35,160 --> 00:35:40,298 AND WE'RE SEPARATING OUT THE 745 00:35:40,298 --> 00:35:41,533 PRECIPITATE ON INNATIVE BLUE 746 00:35:41,533 --> 00:35:43,835 PAGE GEL. 747 00:35:43,835 --> 00:35:48,840 HEALTH CELLS NINJ1 IS A MONOMER 748 00:35:48,840 --> 00:35:49,274 DIMER. 749 00:35:49,274 --> 00:35:54,979 WITHIN ONE HOUR FORMS POLYMERIC 750 00:35:54,979 --> 00:35:56,047 STRUCTURE, RIGHT BANDS, NOT DEAD 751 00:35:56,047 --> 00:35:57,315 IN THE KNOCKOUT. 752 00:35:57,315 --> 00:36:03,054 IF WE LOOK BY IMMUNOSTAINING WE 753 00:36:03,054 --> 00:36:05,457 SEE IN CONTROL CELLS IF I HAVE 754 00:36:05,457 --> 00:36:08,426 USED ON PLASMA MEMBRANE BUT DEAD 755 00:36:08,426 --> 00:36:10,695 CELLS FOLLOWING NIGERICIN 756 00:36:10,695 --> 00:36:14,332 EXPOSURE PUNCTATE FOCI ON CELL 757 00:36:14,332 --> 00:36:14,599 MEMBRANE. 758 00:36:14,599 --> 00:36:16,301 IT'S NOT THERE IN THE NINJ1 759 00:36:16,301 --> 00:36:19,704 KNOCKOUTS, THE STAINING IS 760 00:36:19,704 --> 00:36:20,038 SPECIFIC. 761 00:36:20,038 --> 00:36:22,240 SO, WE WERE SIDETRACKED FOR SOME 762 00:36:22,240 --> 00:36:23,341 TIME BECAUSE HOW DOES THIS 763 00:36:23,341 --> 00:36:23,875 HAPPEN? 764 00:36:23,875 --> 00:36:27,579 HOW DO YOU GO FROM MONOMER TO 765 00:36:27,579 --> 00:36:28,813 POLYMER SO QUICKLY? 766 00:36:28,813 --> 00:36:30,348 THERE MUST BE A SECOND 767 00:36:30,348 --> 00:36:30,949 MESSENGER. 768 00:36:30,949 --> 00:36:35,453 THERE MUST BE A PHOSPHORYLATION, 769 00:36:35,453 --> 00:36:36,454 UBIQUITINATION, SOMETHING MUST 770 00:36:36,454 --> 00:36:36,955 HAVE CHANGED. 771 00:36:36,955 --> 00:36:38,323 AND WE HAVEN'T BEEN ABLE TO 772 00:36:38,323 --> 00:36:39,090 DETECT IT. 773 00:36:39,090 --> 00:36:40,658 I'LL COME BACK TO WHAT OUR 774 00:36:40,658 --> 00:36:43,061 THINKING IS RIGHT NOW. 775 00:36:43,061 --> 00:36:48,500 THERE ISN'T A TRIGGER OF 776 00:36:48,500 --> 00:36:51,703 PROTEOLYTIC CLEAVAGE, 777 00:36:51,703 --> 00:36:56,841 PHOSPHORYLATION, YOUR -- 778 00:36:56,841 --> 00:37:01,112 UBIQUITINATION IS IS 779 00:37:01,112 --> 00:37:01,980 RESPONSIBLE, MONOMER GOING TO 780 00:37:01,980 --> 00:37:02,514 POLYMER. 781 00:37:02,514 --> 00:37:05,383 YOU HAVE TO ASK YOURSELF A VERY 782 00:37:05,383 --> 00:37:06,217 IMPORTANT QUESTION. 783 00:37:06,217 --> 00:37:07,685 WHY WOULD THIS BE COULD BE 784 00:37:07,685 --> 00:37:08,987 SERVED IN EVOLUTIONARY TIME? 785 00:37:08,987 --> 00:37:11,856 BECAUSE WHAT I'M SAYING IS WHAT 786 00:37:11,856 --> 00:37:13,391 WE'VE CONSERVED IS A MECHANISM 787 00:37:13,391 --> 00:37:15,793 THAT MAKES A DEAD CELL DEADER. 788 00:37:15,793 --> 00:37:19,531 THAT DOESN'T MAKE ANY SENSE. 789 00:37:19,531 --> 00:37:21,399 YOU'RE DEAD. 790 00:37:21,399 --> 00:37:23,101 WHY WOULD EVOLUTION CARE? 791 00:37:23,101 --> 00:37:25,303 WELL, I THINK THE REASON FOR 792 00:37:25,303 --> 00:37:28,907 THAT IS THAT AS YOU KNOW, WE 793 00:37:28,907 --> 00:37:30,775 HAVE INJURED AND DEFECTIVE CELLS 794 00:37:30,775 --> 00:37:35,480 ALL THE TIME BEING GENERATED. 795 00:37:35,480 --> 00:37:36,548 EITHER INNOCUOUSLY OR THROUGH 796 00:37:36,548 --> 00:37:42,487 TRAUMA, INEFFECT, ET CETERA. -- 797 00:37:42,487 --> 00:37:46,624 INFECTION, ET CETERA, TAKEN UP 798 00:37:46,624 --> 00:37:48,626 BY ENDOTHELIAL SYSTEM OR 799 00:37:48,626 --> 00:37:50,929 NEIGHBORING CELLS, A BUFFER 800 00:37:50,929 --> 00:37:52,597 TAKES UP INJURED CELLS AND 801 00:37:52,597 --> 00:37:55,166 DIGESTING THEM AND THERE IS NO 802 00:37:55,166 --> 00:37:57,068 INFLAMMATORY RESPONSE. 803 00:37:57,068 --> 00:37:58,336 THINK OF APOPTOTIC CELLS. 804 00:37:58,336 --> 00:38:00,305 BUT IF YOU EXCEED A CERTAIN 805 00:38:00,305 --> 00:38:03,808 THRESHOLD AND THAT THRESHOLD IS 806 00:38:03,808 --> 00:38:11,049 THE ABILITY OF THESE CELLS TO 807 00:38:11,049 --> 00:38:11,816 ENGULF INJURED CELLS, 808 00:38:11,816 --> 00:38:14,218 AFROCYTOSIS, THAT'S A SIGNAL FOR 809 00:38:14,218 --> 00:38:16,854 THE BODY. 810 00:38:16,854 --> 00:38:22,727 IT SAYS WE HAVE INJURY HERE, 811 00:38:22,727 --> 00:38:28,766 BUFFER SYSTEM IS SATURATED, 812 00:38:28,766 --> 00:38:30,068 CELLS RAPIDLY LYSE, RELEASE 813 00:38:30,068 --> 00:38:31,502 MEDIATOR OF INFLAMMATION, THE 814 00:38:31,502 --> 00:38:31,803 DAMPs. 815 00:38:31,803 --> 00:38:33,771 YOU'RE CALLING IN THE IMMUNE 816 00:38:33,771 --> 00:38:36,541 RESPONSE. 817 00:38:36,541 --> 00:38:38,843 SO, THAT'S THE THINKING. 818 00:38:38,843 --> 00:38:40,912 AN EXTENSION OF THAT THINKING IF 819 00:38:40,912 --> 00:38:42,780 YOU'RE ABLE TO INHIBIT LYSIS OF 820 00:38:42,780 --> 00:38:47,485 DEAD CELLS, THAT WOULD 821 00:38:47,485 --> 00:38:48,119 POTENTIALLY BE 822 00:38:48,119 --> 00:38:49,287 ANTI-INFLAMMATORY. 823 00:38:49,287 --> 00:38:51,422 SO I THINK NINJ1 IS CONSERVED IN 824 00:38:51,422 --> 00:38:53,758 EVOLUTION, THERE IN THE FLY, 825 00:38:53,758 --> 00:38:56,160 BECAUSE IT INDUCES RAPID CELL 826 00:38:56,160 --> 00:38:59,330 LYSIS AND THIS SIGNALS, IT'S A 827 00:38:59,330 --> 00:39:03,401 PRIMITIVE WAY TO SIGNAL 828 00:39:03,401 --> 00:39:04,035 INFLAMMATION. 829 00:39:04,035 --> 00:39:05,803 SO A LOT OF THIS IS PUBLISHED, I 830 00:39:05,803 --> 00:39:07,905 WON'T GO INTO IT. 831 00:39:07,905 --> 00:39:09,741 CONSISTENT WITH THIS MODEL IS 832 00:39:09,741 --> 00:39:11,943 THE FACT THAT NINJ1 NULL MICE 833 00:39:11,943 --> 00:39:18,182 ARE SUSCEPTIBLE TO INFECTIONS, 834 00:39:18,182 --> 00:39:18,950 ESPECIALLY INTRACELLULAR 835 00:39:18,950 --> 00:39:21,819 PATHOGENS, RESISTANT TO STERILE 836 00:39:21,819 --> 00:39:22,587 INFLAMMATION. 837 00:39:22,587 --> 00:39:24,255 DATA ON STERILE INFLAMMATION, TO 838 00:39:24,255 --> 00:39:26,424 ADDRESS THIS IN A DEFINITIVE 839 00:39:26,424 --> 00:39:28,960 MANNER WE USED BOTH KNOCKOUT 840 00:39:28,960 --> 00:39:31,362 STUDIES AND WE GENERATED A VERY 841 00:39:31,362 --> 00:39:32,997 POWERFUL TOOL. 842 00:39:32,997 --> 00:39:38,169 WE GENERATED AN ANTIBODY THAT IS 843 00:39:38,169 --> 00:39:43,307 CAPABLE OF NEUTRALIZING NINJ1. 844 00:39:43,307 --> 00:39:48,212 IN ORDER TO DO THAT, WE 845 00:39:48,212 --> 00:39:51,049 EXPRESSED NINJ1 PROTEIN IN 846 00:39:51,049 --> 00:39:51,849 EXTRACELLULAR VESICLES, 847 00:39:51,849 --> 00:39:55,887 GENERATED A LARGE NUMBER OF 848 00:39:55,887 --> 00:39:56,521 RECOMBINANT MONOCLONAL 849 00:39:56,521 --> 00:39:58,322 ANTIBODIES, ASSAYED FOR THEIR 850 00:39:58,322 --> 00:39:59,524 ABILITY TO INHIBIT LDH RELEASE. 851 00:39:59,524 --> 00:40:02,460 THIS IS JUST THE RELEASE OF LDH 852 00:40:02,460 --> 00:40:03,461 ON THE Y-AXIS. 853 00:40:03,461 --> 00:40:05,997 THIS IS THE NINJ1 KNOCKOUT, SO 854 00:40:05,997 --> 00:40:10,568 THERE'S A COME PRO MIGHT HAVE 855 00:40:10,568 --> 00:40:12,437 HAD -- COMPROMISED RELEASE, AND 856 00:40:12,437 --> 00:40:15,807 JUST AS COMPROMISED IS CLONE D1, 857 00:40:15,807 --> 00:40:18,209 IN THE PRESENCE OF D1 YOU 858 00:40:18,209 --> 00:40:20,745 NEUTRALIZE THE ABILITY OF NINJ1 859 00:40:20,745 --> 00:40:23,281 TO LYSE MEMBRANES TO THE SAME 860 00:40:23,281 --> 00:40:31,689 EXTENT AS THE KNOCKOUT. 861 00:40:31,689 --> 00:40:38,463 SO, D1 INHABITS PLASMA MEMBRANE 862 00:40:38,463 --> 00:40:39,030 RUPTURE. 863 00:40:39,030 --> 00:40:45,369 I'LL SHOW YOU DATA, A FUNCTION 864 00:40:45,369 --> 00:40:47,572 OF LPS, UNTREATED CELLS YOU GET 865 00:40:47,572 --> 00:40:50,975 TREMENDOUS RELEASE OF LDH ON 866 00:40:50,975 --> 00:40:53,277 EXPOSURE TO CYTOSOLIC LPS, THE 867 00:40:53,277 --> 00:40:55,680 NINJ1 KNOCKOUT IS THE SOLID 868 00:40:55,680 --> 00:40:58,750 BLACK BAR, PROTECTED, BUT ALMOST 869 00:40:58,750 --> 00:41:01,018 AS WELL PROTECTED IS CELLS 870 00:41:01,018 --> 00:41:03,888 EXPOSED TO THE ANTIBODY ONE 871 00:41:03,888 --> 00:41:05,690 MICROGRAM OR TEN MICROGRAMS PER 872 00:41:05,690 --> 00:41:05,990 MILL. 873 00:41:05,990 --> 00:41:08,926 OKAY. 874 00:41:08,926 --> 00:41:15,466 NOW, HOW THE ANTIBODY WORKS IS 875 00:41:15,466 --> 00:41:22,373 WE BELIEVE IT SUPPRESSES 876 00:41:22,373 --> 00:41:22,740 OLIGOMERIZATION. 877 00:41:22,740 --> 00:41:27,478 IF YOU PURIFY, IT COMES OUT AS A 878 00:41:27,478 --> 00:41:30,515 VERY LARGE MOLECULAR WEIGHT 879 00:41:30,515 --> 00:41:33,251 OBJECT, UNDER SCANNING, UNDER 880 00:41:33,251 --> 00:41:34,552 TRANSMISSION ELECTRON MICROSCOPY 881 00:41:34,552 --> 00:41:37,388 YOU CAN SEE THESE ARE 882 00:41:37,388 --> 00:41:38,923 CHOCK-A-BLOCK FULL OF 883 00:41:38,923 --> 00:41:41,092 POLYMERIZED PROTEINS OFTEN IN 884 00:41:41,092 --> 00:41:42,760 CIRCULAR CONFIGURATIONS, IF YOU 885 00:41:42,760 --> 00:41:45,296 DO THE SAME EXPERIMENT BUT NOW 886 00:41:45,296 --> 00:41:48,132 IN THE PRESENCE OF ANTIBODY, 887 00:41:48,132 --> 00:41:53,738 THEN THERE'S A DEPOLYMERIZATION, 888 00:41:53,738 --> 00:42:01,813 NO EVIDENCE FOR NINJ1 OLIGOMER 889 00:42:01,813 --> 00:42:12,290 FORMATION BY GEL STAINING OR 890 00:42:13,057 --> 00:42:13,391 ELECTROMICROSCOPY. 891 00:42:13,391 --> 00:42:19,463 IN THE U.K. BIOBANK 892 00:42:19,463 --> 00:42:22,233 POLYMORPHISMS IN NINJ1 WERE 893 00:42:22,233 --> 00:42:23,734 ASSOCIATED WITH PEOPLE THAT HAD 894 00:42:23,734 --> 00:42:27,505 LOW LEVELS OF CIRCULATING LIVER 895 00:42:27,505 --> 00:42:31,375 ENZYMES, THAT YOU NORMALLY 896 00:42:31,375 --> 00:42:32,844 MEASURE FOR LIVER DAMAGE. 897 00:42:32,844 --> 00:42:33,744 WE THOUGHT MAYBE LIVER INJURY 898 00:42:33,744 --> 00:42:38,516 WOULD BE A PLACE WE SHOULD LOOK 899 00:42:38,516 --> 00:42:40,017 AT NEUTRALIZING A NINJ1. 900 00:42:40,017 --> 00:42:41,919 SO THE FIRST EXPERIMENT WAS A 901 00:42:41,919 --> 00:42:44,989 GENETIC EXPERIMENT WHERE WE TOOK 902 00:42:44,989 --> 00:42:48,159 OUR NINJ1 KNOCKOUT MICE, AND WE 903 00:42:48,159 --> 00:42:58,636 TREATED THE MICE WITH TNF D 904 00:43:05,710 --> 00:43:05,977 GALACTOSAMINE. 905 00:43:05,977 --> 00:43:07,178 STAINING IS NOT THERE IN THE 906 00:43:07,178 --> 00:43:09,380 KNOCKOUT, IT IS SPECIFIC. 907 00:43:09,380 --> 00:43:10,348 LOOK AT THIS DATA. 908 00:43:10,348 --> 00:43:19,757 IF YOU EXPOSE THE MICE TO TNF 909 00:43:19,757 --> 00:43:22,827 AND GALACTOSAMINE, THEY ARE 910 00:43:22,827 --> 00:43:24,228 GREATLY SUBDUED ALMOST TO 911 00:43:24,228 --> 00:43:31,035 CONTROL LEVELS IN THE NINJ1 912 00:43:31,035 --> 00:43:31,302 KNOCKOUT. 913 00:43:31,302 --> 00:43:34,071 SO, WHAT ABOUT THE ANTIBODY? 914 00:43:34,071 --> 00:43:36,908 NOW, AGAIN, WE USE ANOTHER MODEL 915 00:43:36,908 --> 00:43:37,942 FOR LIVER INJURY. 916 00:43:37,942 --> 00:43:40,678 THIS IS -- WE'RE INJURING THE 917 00:43:40,678 --> 00:43:43,648 LIVER THROUGH CON-A. 918 00:43:43,648 --> 00:43:46,584 THIS RESEMBLES CTL INDUCED 919 00:43:46,584 --> 00:43:47,585 KILLING OF HEPATOCYTES. 920 00:43:47,585 --> 00:43:51,088 AND AGAIN IN THE GENETIC MODEL 921 00:43:51,088 --> 00:43:54,992 YOU SEE SUBSTANTIAL PROTECTION 922 00:43:54,992 --> 00:43:58,029 IN THIS MODEL. 923 00:43:58,029 --> 00:44:00,731 NOW, HERE'S THE EXPERIMENT WITH 924 00:44:00,731 --> 00:44:03,067 THE ANTIBODY, ALBEIT IT IS A 925 00:44:03,067 --> 00:44:04,702 PRE-TREATMENT REGIMEN, SO IT'S A 926 00:44:04,702 --> 00:44:06,370 RELATIVELY LOW BAR, SO TAKE IT 927 00:44:06,370 --> 00:44:07,872 FOR WHAT IT'S WORTH. 928 00:44:07,872 --> 00:44:10,741 WE GIVE THE ANTIBODY FIRST AND 929 00:44:10,741 --> 00:44:14,245 THEN GIVE THE CON-A. 930 00:44:14,245 --> 00:44:16,314 I HOPE YOU'LL REALIZE WHEN YOU 931 00:44:16,314 --> 00:44:18,182 LOOK AT LDH OR LIVER ENZYMES, 932 00:44:18,182 --> 00:44:20,251 THAT IN THE PRESENCE OF THE 933 00:44:20,251 --> 00:44:23,421 ANTIBODY SHOWN HERE IN BLUE 934 00:44:23,421 --> 00:44:24,722 THERE'S A DRAMATIC DIFFERENCE, 935 00:44:24,722 --> 00:44:27,692 DRAMATIC SUPPRESSION OF LIVER 936 00:44:27,692 --> 00:44:28,059 INJURY. 937 00:44:28,059 --> 00:44:33,698 THIS IS CORROBORATED BY RELEASE 938 00:44:33,698 --> 00:44:35,766 OF CYTOKINES AND SIMPLY JUST 939 00:44:35,766 --> 00:44:38,636 STAINING THE LIVER FOR 940 00:44:38,636 --> 00:44:39,170 NEUTROPHILS. 941 00:44:39,170 --> 00:44:42,440 SO YOU SUPPRESS IMMUNE INJURY. 942 00:44:42,440 --> 00:44:45,042 943 00:44:45,042 --> 00:44:47,712 SO, THIS SUGGESTS THAT NINJ1 IS 944 00:44:47,712 --> 00:44:51,215 POTENTIALLY A DRUGGABLE TARGET. 945 00:44:51,215 --> 00:44:55,252 AND IT BRINGS TO THE FORE THE 946 00:44:55,252 --> 00:44:59,090 CONCEPT THAT WHEN YOU HAVE 947 00:44:59,090 --> 00:45:01,058 OVERWHELMING DEATH OF CELLS, 948 00:45:01,058 --> 00:45:04,228 THAT THE PHAGOCYTIC SYSTEM CAN 949 00:45:04,228 --> 00:45:08,299 NO LONGER DEAL WITH EXCESS DEAD 950 00:45:08,299 --> 00:45:09,700 CELLS, RAPID LYSIS MEDIATED BY 951 00:45:09,700 --> 00:45:13,671 NINJ1, LEADS TO THE RELEASE OF 952 00:45:13,671 --> 00:45:15,840 DAMPs, AND INITIATION OF 953 00:45:15,840 --> 00:45:16,974 TISSUE INFLAMMATION. 954 00:45:16,974 --> 00:45:20,444 AND THAT THIS INJURY IN CASES OF 955 00:45:20,444 --> 00:45:24,048 STERILE INFLAMMATION COULD BE 956 00:45:24,048 --> 00:45:25,349 BENEFICIALLY BLOCKED BY 957 00:45:25,349 --> 00:45:32,590 NEUTRALIZING NINJ1 WITH AN 958 00:45:32,590 --> 00:45:34,358 ANTIBODY TO NINJ1. 959 00:45:34,358 --> 00:45:39,263 BUT WHERE FROM HERE? 960 00:45:39,263 --> 00:45:41,999 HOW IS NINJ1 GETTING ACTIVATED, 961 00:45:41,999 --> 00:45:42,666 $64,000 QUESTION. 962 00:45:42,666 --> 00:45:47,972 NOW, I DON'T THINK IT'S A 963 00:45:47,972 --> 00:45:48,939 POST-TRANSLATIONAL MODIFICATION. 964 00:45:48,939 --> 00:45:51,776 THERE'S SO MANY ROADS TO NINJ1 965 00:45:51,776 --> 00:45:54,378 ACTIVATION, WHETHER YOU KILL THE 966 00:45:54,378 --> 00:45:58,682 CELLS WITH CISPLATIN OR 967 00:45:58,682 --> 00:45:59,884 LISTERIA, VERY LITTLE IN COMMON 968 00:45:59,884 --> 00:46:02,686 BETWEEN WHAT'S DOING THE 969 00:46:02,686 --> 00:46:02,920 KILLING. 970 00:46:02,920 --> 00:46:07,324 I THINK NINJ1 IS MEASURING, 971 00:46:07,324 --> 00:46:08,959 RESPONDING TO THE HEALTH OF THE 972 00:46:08,959 --> 00:46:10,161 MEMBRANE. 973 00:46:10,161 --> 00:46:12,163 NOW, THE QUESTION IS, IS IT 974 00:46:12,163 --> 00:46:15,866 CHANGES IN MEMBRANE TENSION OR 975 00:46:15,866 --> 00:46:20,471 MEMBRANE COMPOSITION, OR THE 976 00:46:20,471 --> 00:46:21,572 BINDING, OR ABSENCE OF PARTNER, 977 00:46:21,572 --> 00:46:25,076 WHAT IS IT THAT IS PRECISELY 978 00:46:25,076 --> 00:46:25,376 RESPONSIBLE? 979 00:46:25,376 --> 00:46:29,346 AND I THINK THIS IS THE QUESTION 980 00:46:29,346 --> 00:46:31,949 I WOULD BRING YOUR ATTENTION TO 981 00:46:31,949 --> 00:46:36,887 BECAUSE IT'S BEGINNING TO BE 982 00:46:36,887 --> 00:46:37,254 ANSWERED. 983 00:46:37,254 --> 00:46:39,623 NOW, THE FIRST ATTEMPT AT THAT 984 00:46:39,623 --> 00:46:42,026 WAS TO -- WHICH WAS VERY 985 00:46:42,026 --> 00:46:45,529 GRATIFYING FOR US BECAUSE WE 986 00:46:45,529 --> 00:46:50,801 WERE ON A LIMB SAYING NINJ1 987 00:46:50,801 --> 00:46:53,070 FORMS MEGA PORES, INVOLVED WITH 988 00:46:53,070 --> 00:47:02,847 CELL LYSIS, A PAPER THAT DID 989 00:47:02,847 --> 00:47:06,317 STRUCTURE OF ACTIVATED 990 00:47:06,317 --> 00:47:08,285 POLYMERIC NINJ1, THERE ARE 991 00:47:08,285 --> 00:47:18,596 PAPERS IN PRESS FROM WU AND ZING 992 00:47:18,596 --> 00:47:20,030 HONG DAI WITH SIMILAR 993 00:47:20,030 --> 00:47:20,331 CONCLUSIONS. 994 00:47:20,331 --> 00:47:22,766 GENERALLY THEY SAY THIS. 995 00:47:22,766 --> 00:47:24,969 IN THE ACTIVE POLYMERIC STATE, 996 00:47:24,969 --> 00:47:28,272 THE ARCHITECTURE OF NINJ1 IS SO. 997 00:47:28,272 --> 00:47:32,943 YOU HAVE TWO TRANSMEMBRANE 998 00:47:32,943 --> 00:47:36,847 HELICES I TALKED ABOUT, BUT THE 999 00:47:36,847 --> 00:47:39,817 AMPIPATHIC IS EMBEDDED IN THE 1000 00:47:39,817 --> 00:47:42,920 MEMBRANE, ACTUALLY TWO HELICES. 1001 00:47:42,920 --> 00:47:46,190 THERE'S A KINK. 1002 00:47:46,190 --> 00:47:49,393 THIS KINK IS IMPORTANT BECAUSE 1003 00:47:49,393 --> 00:47:52,997 THE KINKED HELIX ACTS AS A 1004 00:47:52,997 --> 00:47:55,466 CROSS-BRACE BECAUSE IT INTERACTS 1005 00:47:55,466 --> 00:47:56,400 CAN A JOINT TRANSMEMBRANE 1006 00:47:56,400 --> 00:48:00,437 DOMAIN, YOU CAN SEE HOW IT COULD 1007 00:48:00,437 --> 00:48:01,472 TRIGGER POLYMERIZATION. 1008 00:48:01,472 --> 00:48:07,645 SO IT HAS THE APPROPRIATE 1009 00:48:07,645 --> 00:48:10,181 AMPIPATHIC CHARACTER, AND COULD 1010 00:48:10,181 --> 00:48:13,884 POTENTIALLY GENERATE VERY LARGE 1011 00:48:13,884 --> 00:48:15,319 PORES, THEY ESTIMATE 1012 00:48:15,319 --> 00:48:17,621 500-NANOMETER PORES ESSENTIALLY 1013 00:48:17,621 --> 00:48:18,389 FOR MEMBRANE LYSIS. 1014 00:48:18,389 --> 00:48:22,960 SO I THINK THIS IS AN IMPORTANT 1015 00:48:22,960 --> 00:48:24,628 STEP, CERTAINLY DOESN'T ANSWER 1016 00:48:24,628 --> 00:48:25,496 THE QUESTION. 1017 00:48:25,496 --> 00:48:29,433 AND WHAT WE'RE SORT OF PRESENTLY 1018 00:48:29,433 --> 00:48:31,202 CONCERNED WITH IS THAT HOW SURE 1019 00:48:31,202 --> 00:48:36,874 ARE WE THAT THE N-TERMINUS IS 1020 00:48:36,874 --> 00:48:37,208 OUTSIDE? 1021 00:48:37,208 --> 00:48:39,944 BECAUSE THIS IS ONE MODEL, THE 1022 00:48:39,944 --> 00:48:41,045 N-TERMINUS IS OUTSIDE. 1023 00:48:41,045 --> 00:48:46,383 SO WE WOULD SAY IN THE INACTIVE 1024 00:48:46,383 --> 00:48:50,120 MONOMERIC STATE, THE N-TERMINUS 1025 00:48:50,120 --> 00:48:50,988 IS OUTSIDE. 1026 00:48:50,988 --> 00:48:51,989 THAT'S THIS. 1027 00:48:51,989 --> 00:48:56,594 BUT IN THE ACTIVE STATE IT'S 1028 00:48:56,594 --> 00:48:57,661 INSIDE, AND IT'S KINKED. 1029 00:48:57,661 --> 00:49:02,032 THAT'S A HUGE CLAIM TO MAKE. 1030 00:49:02,032 --> 00:49:02,533 ALMOST THERMODYNAMICALLY 1031 00:49:02,533 --> 00:49:03,567 IMPOSSIBLE FOR THIS TO HAPPEN. 1032 00:49:03,567 --> 00:49:05,336 I DON'T HAVE THE ANSWER BUT I'LL 1033 00:49:05,336 --> 00:49:07,204 SHOW YOU THE EVIDENCE. 1034 00:49:07,204 --> 00:49:10,241 WE BELIEVE THAT AT LEAST A 1035 00:49:10,241 --> 00:49:12,643 FRACTION OF NINJ1 HAS THIS 1036 00:49:12,643 --> 00:49:13,744 ORIENTATION WITH THE N-TERMINUS 1037 00:49:13,744 --> 00:49:15,613 AMPIPATHIC ON THE OUTSIDE, AND 1038 00:49:15,613 --> 00:49:17,581 MAY BE THAT IT'S JUST HUGGING 1039 00:49:17,581 --> 00:49:18,949 YOU THE PLASMA MEMBRANE, 1040 00:49:18,949 --> 00:49:23,053 PARTIALLY EMBEDDED IN THE PLASMA 1041 00:49:23,053 --> 00:49:23,554 MEMBRANE. 1042 00:49:23,554 --> 00:49:26,056 WHEN WE DO FAX WITH CLONE 25 WE 1043 00:49:26,056 --> 00:49:29,193 SEE A SIGNAL. 1044 00:49:29,193 --> 00:49:39,637 IF WE C-TERMINUS, RETAINED, 1045 00:49:39,637 --> 00:49:42,006 N-TERMINUS IT'S GONE. 1046 00:49:42,006 --> 00:49:44,708 THERE ARE OTHER INTERPRETATIONS, 1047 00:49:44,708 --> 00:49:47,511 OPEN WHERE WE'RE AT RIGHT NOW. 1048 00:49:47,511 --> 00:49:50,581 SO WE'RE AT THE VERY CUSP OF 1049 00:49:50,581 --> 00:49:51,649 HAVING SOLVED THE MONOMERIC 1050 00:49:51,649 --> 00:49:53,617 STRUCTURE, AND I'M HOPING THAT 1051 00:49:53,617 --> 00:49:56,687 IN THE NEXT WEEK OR TWO WE'LL BE 1052 00:49:56,687 --> 00:50:01,859 ABLE TO GET BETTER INSIGHT INTO 1053 00:50:01,859 --> 00:50:02,926 MOLECULAR GYMNASTICS BUT IT 1054 00:50:02,926 --> 00:50:04,461 WON'T ADDRESS THE QUESTION OF 1055 00:50:04,461 --> 00:50:05,663 WHAT TRIGGERS IT. 1056 00:50:05,663 --> 00:50:07,631 IS IT TENSION, IS IT 1057 00:50:07,631 --> 00:50:09,366 COMPOSITION, WHAT IS IT? 1058 00:50:09,366 --> 00:50:10,934 AND I THINK THAT WILL BE THE 1059 00:50:10,934 --> 00:50:15,606 SUBJECT OF A LOT OF PAPERS BY 1060 00:50:15,606 --> 00:50:16,407 OTHERS AND HOPEFULLY BY 1061 00:50:16,407 --> 00:50:19,610 OURSELVES. 1062 00:50:19,610 --> 00:50:21,078 1063 00:50:21,078 --> 00:50:24,481 SO, TWO CONCLUSIONS FROM THE 1064 00:50:24,481 --> 00:50:25,883 TALK. 1065 00:50:25,883 --> 00:50:27,551 LEADERLESS CYTOKINES ARE 1066 00:50:27,551 --> 00:50:30,054 RELEASED BY GASDERMIN-D PORES. 1067 00:50:30,054 --> 00:50:33,524 AND THE SECOND CONCLUSION, VERY 1068 00:50:33,524 --> 00:50:34,224 SURPRISING, THAT CELL LYSIS 1069 00:50:34,224 --> 00:50:38,262 FOLLOWING CELL DEATH IS NOT A 1070 00:50:38,262 --> 00:50:39,797 PASSIVE EVENT, LIKE MISS JONES 1071 00:50:39,797 --> 00:50:43,434 TAUGHT US IN HIGH SCHOOL, THAT'S 1072 00:50:43,434 --> 00:50:47,504 ONE GREATLY ACCELERATED BY 1073 00:50:47,504 --> 00:50:48,605 NINJ1. 1074 00:50:48,605 --> 00:50:48,806 OKAY. 1075 00:50:48,806 --> 00:50:49,573 I'LL END THERE. 1076 00:50:49,573 --> 00:50:52,009 HOW AM I DOING FOR TIME? 1077 00:50:52,009 --> 00:50:52,743 ALL RIGHT. 1078 00:50:52,743 --> 00:50:56,313 SO THE WORK HAS BEEN ONGOING, 1079 00:50:56,313 --> 00:51:00,117 PLENTY OF COLLABORATION WITH MY 1080 00:51:00,117 --> 00:51:03,087 COLLEAGUE KAYAGAKI AND HIS 1081 00:51:03,087 --> 00:51:05,622 GROUP, STOWE AND LEE, OPHER WAS 1082 00:51:05,622 --> 00:51:07,691 INSTRUMENTAL IN DISCOVERY OF 1083 00:51:07,691 --> 00:51:08,258 NINJ1. 1084 00:51:08,258 --> 00:51:11,195 AND WE'VE HAD THE FORWARD 1085 00:51:11,195 --> 00:51:14,064 GENETIC SCREEN, MASSIVE EFFORT 1086 00:51:14,064 --> 00:51:21,038 DONE IN COLLABORATION WITH ANU, 1087 00:51:21,038 --> 00:51:21,739 CANBERRA. 1088 00:51:21,739 --> 00:51:29,346 STRUCTURAL WORK DONE BY ISHAN 1089 00:51:29,346 --> 00:51:32,649 AND KAMELA, RYAN AND LEE AND 1090 00:51:32,649 --> 00:51:34,918 PATHOLOGY BY JOSH WEBSTER. 1091 00:51:34,918 --> 00:51:36,453 CHRIS GOODNOW IS PART OF THE 1092 00:51:36,453 --> 00:51:37,888 CANBERRA GROUP BUT HAS SINCE 1093 00:51:37,888 --> 00:51:39,423 MOVED TO SYDNEY. 1094 00:51:39,423 --> 00:51:40,391 THE WORK WOULDN'T HAVE BEEN 1095 00:51:40,391 --> 00:51:42,693 POSSIBLE WITHOUT THEM. 1096 00:51:42,693 --> 00:51:45,429 I HOPE I'VE TAKEN YOU ON A 1097 00:51:45,429 --> 00:51:46,764 PERSONAL JOURNEY OF DISCOVERY. 1098 00:51:46,764 --> 00:51:49,466 I DIDN'T SHOW SIGNAL CELL 1099 00:51:49,466 --> 00:51:51,435 RNAseq, DID I? 1100 00:51:51,435 --> 00:51:53,537 WELL, I'LL OLD WORLD, WHAT CAN I 1101 00:51:53,537 --> 00:51:53,704 SAY? 1102 00:51:53,704 --> 00:51:53,971 THANK YOU. 1103 00:51:53,971 --> 00:51:56,473 [APPLAUSE] 1104 00:51:56,473 --> 00:52:02,513 1105 00:52:02,513 --> 00:52:09,520 1106 00:52:09,520 --> 00:52:11,488 >> GREAT TALK. 1107 00:52:11,488 --> 00:52:12,589 I APPRECIATED ALL THE WESTERN 1108 00:52:12,589 --> 00:52:14,024 BLOTS OF COURSE. 1109 00:52:14,024 --> 00:52:17,628 I WAS WONDERING, TWO QUESTIONS. 1110 00:52:17,628 --> 00:52:19,596 ONE, SO CELL DEATH, APOPTOSIS IS 1111 00:52:19,596 --> 00:52:21,331 ALSO IMPORTANT DURING 1112 00:52:21,331 --> 00:52:21,632 DEVELOPMENT. 1113 00:52:21,632 --> 00:52:22,866 FROM YOUR MOUSE MODELS AND SO ON 1114 00:52:22,866 --> 00:52:24,935 DO YOU HAVE ANY INDICATION THAT 1115 00:52:24,935 --> 00:52:29,006 NINJ1 COULD ALSO PLAY A ROLE 1116 00:52:29,006 --> 00:52:32,509 THERE? 1117 00:52:32,509 --> 00:52:35,345 DO YOU SEE RATIOS? 1118 00:52:35,345 --> 00:52:37,514 TO THE MECHANISM I HOPE I DIDN'T 1119 00:52:37,514 --> 00:52:40,584 MISS IT, BUT YOU HAVE ANOTHER 1120 00:52:40,584 --> 00:52:41,552 MONOCLONAL ANTIBODY, CLONE 25, 1121 00:52:41,552 --> 00:52:43,454 DID YOU ALSO TEST THAT? 1122 00:52:43,454 --> 00:52:48,792 DOES THAT ALSO INHIBIT THE 1123 00:52:48,792 --> 00:52:49,159 OLIGOMERIZATION? 1124 00:52:49,159 --> 00:52:51,895 >> CLONE 25 DOESN'T WORK AT AS 1125 00:52:51,895 --> 00:52:52,996 WELL, PARTIAL INHIBITOR, THAT'S 1126 00:52:52,996 --> 00:52:57,367 WHY WE WENT TO D1. 1127 00:52:57,367 --> 00:53:01,071 AND YOUR FIRST QUESTION WAS -- 1128 00:53:01,071 --> 00:53:02,840 >> IS THERE ANY WAY THAT NINJ1 1129 00:53:02,840 --> 00:53:04,641 COULD ALSO PLAY A ROLE IN -- 1130 00:53:04,641 --> 00:53:07,144 >> OH, YEAH, IN DEVELOPMENT. 1131 00:53:07,144 --> 00:53:14,117 YOU KNOW, I THINK NINJ1 COMES 1132 00:53:14,117 --> 00:53:17,087 INTO EFFECT WHEN DEATH IS 1133 00:53:17,087 --> 00:53:19,389 OVERWHELMING, SO NORMALLY IN 1134 00:53:19,389 --> 00:53:20,591 DEVELOPMENTAL DEATH, THE 1135 00:53:20,591 --> 00:53:22,125 SURROUNDING CELLS WILL ENGULF 1136 00:53:22,125 --> 00:53:22,593 IT. 1137 00:53:22,593 --> 00:53:27,364 YOU KNOW, A SLAUGHTERHOUSE IS 1138 00:53:27,364 --> 00:53:31,001 99.9% OF CELLS ARE GOING TO 1139 00:53:31,001 --> 00:53:33,504 UNDERGO APOPTOTIC DEMISE. 1140 00:53:33,504 --> 00:53:36,573 IF YOU STAINED FOR CLEAVED 1141 00:53:36,573 --> 00:53:38,108 CASPASE 3, YOU SEE VERY FEW, 1142 00:53:38,108 --> 00:53:39,643 IT'S SO RAPID. 1143 00:53:39,643 --> 00:53:41,612 I TELL PEOPLE THERE ARE A 1144 00:53:41,612 --> 00:53:46,316 MILLION CELLS THAT UNDERGO APOPS 1145 00:53:46,316 --> 00:53:47,417 APOPTOSIS EVERY SECOND, THAT 1146 00:53:47,417 --> 00:53:47,951 DOESN'T RESONATE. 1147 00:53:47,951 --> 00:53:50,921 THIS IS THE NUMBER OF CELLS THAT 1148 00:53:50,921 --> 00:53:52,789 UNDERGOES APOPTOSIS, IT'S TWO 1149 00:53:52,789 --> 00:53:54,091 EGGS WORTH OF APOPTOSISU HAPPENS 1150 00:53:54,091 --> 00:53:55,726 IN YOU EVERY DAY. 1151 00:53:55,726 --> 00:53:59,763 YOU'RE NOT INFLAMED. 1152 00:53:59,763 --> 00:54:03,267 THAT IS BECAUSE YOUR SYSTEM OF 1153 00:54:03,267 --> 00:54:06,236 CLEARING IS THAT EFFICIENT. 1154 00:54:06,236 --> 00:54:13,010 BUT WHEN YOU HAVE OVERWHELMING 1155 00:54:13,010 --> 00:54:17,414 INJURY, SYSTEM SATURATED, YOU 1156 00:54:17,414 --> 00:54:20,150 GET LYSIS AND SIGNALING. 1157 00:54:20,150 --> 00:54:21,685 >> BEAUTIFUL, WONDERFUL WORK. 1158 00:54:21,685 --> 00:54:23,420 I HAVE A QUESTION. 1159 00:54:23,420 --> 00:54:25,489 MAYBE I MISSED SOMETHING BECAUSE 1160 00:54:25,489 --> 00:54:27,691 YOU MENTIONED THAT THIS NINJ1 1161 00:54:27,691 --> 00:54:33,497 MAYBE THE LAST STEP TO CLEAR 1162 00:54:33,497 --> 00:54:37,868 WHEN CELLS ARE INJURED OR 1163 00:54:37,868 --> 00:54:40,370 APOPTOTIC. 1164 00:54:40,370 --> 00:54:45,842 WE KNOW IF THE CELLS BETWEEN 1165 00:54:45,842 --> 00:54:53,850 INJURED OR NECROTIC CELLS GET 1166 00:54:53,850 --> 00:54:55,252 QUITE DIFFERENT OUTCOME. 1167 00:54:55,252 --> 00:54:56,687 MY QUESTION IS DO YOU HAVE 1168 00:54:56,687 --> 00:54:58,889 ANY -- MAYBE I MISSED SOMETHING. 1169 00:54:58,889 --> 00:55:00,090 >> NO, YOU DIDN'T MISS 1170 00:55:00,090 --> 00:55:00,424 SOMETHING. 1171 00:55:00,424 --> 00:55:01,491 YOU'RE RIGHT. 1172 00:55:01,491 --> 00:55:05,462 I WANT TO MAKE ANOTHER IMPORTANT 1173 00:55:05,462 --> 00:55:07,431 POINT, EMPHASIZED IN THE REVIEW 1174 00:55:07,431 --> 00:55:08,932 CELL DEATH PUBLISHED TWO MONTHS 1175 00:55:08,932 --> 00:55:09,099 AGO. 1176 00:55:09,099 --> 00:55:12,135 SO HAVE A LOOK AT THAT. 1177 00:55:12,135 --> 00:55:13,103 IF YOU HAVE OVERWHELMING 1178 00:55:13,103 --> 00:55:15,205 APOPTOSIS YOU WILL HAVE 1179 00:55:15,205 --> 00:55:16,506 SECONDARY NECROSIS IN AN 1180 00:55:16,506 --> 00:55:17,441 ORGANISM. 1181 00:55:17,441 --> 00:55:19,676 SO WHILE WE TRY TO TEND TO THINK 1182 00:55:19,676 --> 00:55:22,179 OF APOPTOTIC DEATH AS 1183 00:55:22,179 --> 00:55:25,048 IMMUNOLOGICALLY SILENT, OVER A 1184 00:55:25,048 --> 00:55:27,117 CERTAIN THRESHOLD, IT WILL 1185 00:55:27,117 --> 00:55:28,218 INDUCE INFLAMMATION BECAUSE 1186 00:55:28,218 --> 00:55:30,187 THOSE APOPTOTIC CELLS WILL 1187 00:55:30,187 --> 00:55:31,154 UNDERGO SECONDARY NECROSIS BUT 1188 00:55:31,154 --> 00:55:33,724 YOU HAVE TO HAVE OVERWHELMING 1189 00:55:33,724 --> 00:55:39,596 APOPTOSIS. 1190 00:55:39,596 --> 00:55:40,430 >> HI. 1191 00:55:40,430 --> 00:55:41,898 WHEN YOU THINK ABOUT LIKEth 1192 00:55:41,898 --> 00:55:45,636 THE SPEED OF THE PROCESS, LIKE 1193 00:55:45,636 --> 00:55:48,271 YOU SUGGEST THAT ONE, THERE'S AN 1194 00:55:48,271 --> 00:55:48,905 INACTIVE AND ACTIVE STRUCTURE, 1195 00:55:48,905 --> 00:55:53,744 THIS IS LIKE THE MECHANISM WHICH 1196 00:55:53,744 --> 00:55:55,479 RELEASES PORES, MEGA PORES, IF I 1197 00:55:55,479 --> 00:55:56,680 GET IT RIGHT. 1198 00:55:56,680 --> 00:56:01,251 LIKE WHAT HAPPENS, SO WHERE ARE 1199 00:56:01,251 --> 00:56:02,486 THOSE MOLECULES LOCALIZED? 1200 00:56:02,486 --> 00:56:04,655 ALWAYS THEY MEMBRANE AND THEN 1201 00:56:04,655 --> 00:56:06,423 THEY JUST GET BROUGHT TOGETHER 1202 00:56:06,423 --> 00:56:10,894 OR ARE THEY IN INTRACELLULAR 1203 00:56:10,894 --> 00:56:13,964 COMPARTMENTS TRANSFERRED TO 1204 00:56:13,964 --> 00:56:14,231 MEMBRANES? 1205 00:56:14,231 --> 00:56:16,166 >> VERY GOOD QUESTION. 1206 00:56:16,166 --> 00:56:20,237 SO, THERE'S A HUGE RESIDENT POOL 1207 00:56:20,237 --> 00:56:21,304 AT THE MEMBRANE. 1208 00:56:21,304 --> 00:56:26,009 BUT WHEN WE DO OUR STAINING, IT 1209 00:56:26,009 --> 00:56:27,344 IS CERTAINLY PRESENT IN WHAT 1210 00:56:27,344 --> 00:56:30,380 SEEMS TO BE THE SORT OF ER 1211 00:56:30,380 --> 00:56:30,714 GOLGI. 1212 00:56:30,714 --> 00:56:35,485 I DON'T KNOW WHETHER THAT'S JUST 1213 00:56:35,485 --> 00:56:40,624 NINJ1 ON ITS WAY OR IF THERE'S 1214 00:56:40,624 --> 00:56:42,225 NINJ1 RESIDENT THERE. 1215 00:56:42,225 --> 00:56:45,262 BUT I WOULD VENTURE TO SAY THAT 1216 00:56:45,262 --> 00:56:48,365 IT'S THE PLASMA MEMBRANE NINJ1 1217 00:56:48,365 --> 00:56:50,434 THAT IS RESPONSIBLE FOR THIS 1218 00:56:50,434 --> 00:56:52,636 MEGA PORE FORMATION THAT YOU 1219 00:56:52,636 --> 00:56:52,803 SEE. 1220 00:56:52,803 --> 00:56:56,707 NINJ1 IS NOT, FOR EXAMPLE, ON 1221 00:56:56,707 --> 00:56:58,308 MITOCHONDRIAL MEMBRANES. 1222 00:56:58,308 --> 00:56:59,710 >> DO YOU THINK THAT THERE IS 1223 00:56:59,710 --> 00:57:02,345 LIKE REQUIREMENT FOR THE 1224 00:57:02,345 --> 00:57:03,380 TRANSLOCATION FROM INTRACELLULAR 1225 00:57:03,380 --> 00:57:04,648 MEMBRANES TO FORM THESE PORES OR 1226 00:57:04,648 --> 00:57:08,452 DO YOU THINK IT'S SIMPLY LIKE 1227 00:57:08,452 --> 00:57:09,886 ARE THERE LOCATIONS OR 1228 00:57:09,886 --> 00:57:10,954 PSEUDOLOCATIONS IN MEMBRANES? 1229 00:57:10,954 --> 00:57:14,391 >> YEAH, NO, NO, I THINK IT'S 1230 00:57:14,391 --> 00:57:16,560 ALL IN THE PLASMA MEMBRANE. 1231 00:57:16,560 --> 00:57:17,761 THEY ARE MONITORING THE HEALTH. 1232 00:57:17,761 --> 00:57:18,361 THINK OF A MECHANISM, I DON'T 1233 00:57:18,361 --> 00:57:20,731 KNOW IF THIS IS THE CASE, BUT WE 1234 00:57:20,731 --> 00:57:23,467 KNOW THAT ALL DYING CELLS WILL 1235 00:57:23,467 --> 00:57:25,102 LOSE ATP, RIGHT? 1236 00:57:25,102 --> 00:57:27,838 AND WE KNOW TO MAINTAIN 1237 00:57:27,838 --> 00:57:31,875 ASYMMETRY ARE YOU HAVE TO EXPEND 1238 00:57:31,875 --> 00:57:32,442 ENERGY. 1239 00:57:32,442 --> 00:57:33,510 YOU CAN IMAGINE DYING CELLS ONE 1240 00:57:33,510 --> 00:57:36,480 THING THEY WILL HAVE IN COMMON 1241 00:57:36,480 --> 00:57:39,316 IS ALTERATION OF THEIR MEMBRANE 1242 00:57:39,316 --> 00:57:39,950 COMPOSITION. 1243 00:57:39,950 --> 00:57:40,183 RIGHT? 1244 00:57:40,183 --> 00:57:41,918 MAYBE IT MONITORS THAT. 1245 00:57:41,918 --> 00:57:43,153 BUT IT'S -- I DON'T THINK IT'S 1246 00:57:43,153 --> 00:57:45,655 THE ONE THAT IS TRANSLOCATED. 1247 00:57:45,655 --> 00:57:47,991 I THINK IT'S THE ONE THAT'S 1248 00:57:47,991 --> 00:57:49,926 ALREADY PRESENT ON THE PLASMA 1249 00:57:49,926 --> 00:57:52,596 MEMBRANE. 1250 00:57:52,596 --> 00:57:54,798 >> SO THEN DO YOU SEE 1251 00:57:54,798 --> 00:57:58,368 DEPOLARIZATION OF THE MEMBRANES? 1252 00:57:58,368 --> 00:58:01,471 DO YOU SEE LIKE PHOSPHOLIPIDS 1253 00:58:01,471 --> 00:58:02,372 SHIFT FROM ENTRY OUTSIDE OR DID 1254 00:58:02,372 --> 00:58:07,043 YOU SEE CHANGES IN THE KNOCKOUT 1255 00:58:07,043 --> 00:58:07,644 MICE? 1256 00:58:07,644 --> 00:58:09,880 >> NO, OH, YEAH, IT'S NOT AS 1257 00:58:09,880 --> 00:58:11,214 SIMPLE AS THAT. 1258 00:58:11,214 --> 00:58:12,516 BUT, YEAH, NO, NO, YOU'RE 1259 00:58:12,516 --> 00:58:14,050 GETTING AT THE RIGHT ANGLE. 1260 00:58:14,050 --> 00:58:15,919 I THINK THERE WILL BE A PAPER 1261 00:58:15,919 --> 00:58:18,421 COMING OUT SOON, NOT FROM US, 1262 00:58:18,421 --> 00:58:20,056 FROM SOMEBODY ELSE, SAYING THAT 1263 00:58:20,056 --> 00:58:21,258 IT'S CALCIUM FLUX THAT LEADS US. 1264 00:58:21,258 --> 00:58:23,059 I DON'T KNOW. 1265 00:58:23,059 --> 00:58:25,529 WE DON'T SEE THAT. 1266 00:58:25,529 --> 00:58:26,429 WHO KNOWS. 1267 00:58:26,429 --> 00:58:32,702 IT IS A CHANGE IN THE MEMBRANE. 1268 00:58:32,702 --> 00:58:33,003 YEAH? 1269 00:58:33,003 --> 00:58:34,971 >> FIRST, REALLY EXCITING WORK. 1270 00:58:34,971 --> 00:58:35,972 EXCELLENT TALK. 1271 00:58:35,972 --> 00:58:38,575 >> NO SINGLE CELL RNAseq? 1272 00:58:38,575 --> 00:58:39,776 NO. 1273 00:58:39,776 --> 00:58:40,177 [LAUGHTER] 1274 00:58:40,177 --> 00:58:40,377 DAMN! 1275 00:58:40,377 --> 00:58:43,680 >> I DID HAVE TWO QUESTIONS. 1276 00:58:43,680 --> 00:58:45,348 THE FIRST ONE, ON THE MOUSE 1277 00:58:45,348 --> 00:58:50,954 MODEL OF THE LIVER, AND LOWER 1278 00:58:50,954 --> 00:58:53,390 ALT AND ASC DID YOU ALSO MEASURE 1279 00:58:53,390 --> 00:58:55,692 LIVER FUNCTION OR DEATH? 1280 00:58:55,692 --> 00:58:58,295 ASC AND ALT WILL BE RELEASED AS 1281 00:58:58,295 --> 00:59:04,768 CELL EXPLODES, YOU'RE PREVENTING 1282 00:59:04,768 --> 00:59:08,371 THAT, COULD THEY JUST HAVE BAD 1283 00:59:08,371 --> 00:59:08,905 LIVES? 1284 00:59:08,905 --> 00:59:11,541 >> WE'VE RUN A SURVIVAL ASSAY 1285 00:59:11,541 --> 00:59:13,276 AND WE'VE SEEN A SURVIVAL 1286 00:59:13,276 --> 00:59:14,678 BENEFIT IN THESE EXPERIMENTS 1287 00:59:14,678 --> 00:59:18,114 IT'S DIFFICULT TO SEE ONE 1288 00:59:18,114 --> 00:59:20,717 BECAUSE THE STIMULUS IS, WITH 1289 00:59:20,717 --> 00:59:21,484 TIME, JUST LETHAL. 1290 00:59:21,484 --> 00:59:25,088 I THINK WHEN YOU HAVE SUCH 1291 00:59:25,088 --> 00:59:27,290 LYSIS, INTENSE LYSIS, LIKE IN 1292 00:59:27,290 --> 00:59:30,894 THESE MODELS, YOU GET MASSIVE 1293 00:59:30,894 --> 00:59:34,064 ELECTROLYTE IMBALANCES, PROBABLY 1294 00:59:34,064 --> 00:59:35,599 LIKE HYPER KULEMUA, OTHER 1295 00:59:35,599 --> 00:59:42,272 ASSOCIATED FACTORS, WE DO SEE A 1296 00:59:42,272 --> 00:59:42,939 SURVIVAL BENEFIT. 1297 00:59:42,939 --> 00:59:44,908 >> I'VE SEEN ONES LOOKING AT THE 1298 00:59:44,908 --> 00:59:47,277 RECOVERY RATE OF LIKE IF YOU 1299 00:59:47,277 --> 00:59:52,148 HAVE ACUTE NECROSIS OF THE 1300 00:59:52,148 --> 00:59:54,985 KIDNEY FIT GOES THROUGH 1301 00:59:54,985 --> 00:59:57,587 APOPTOSIS IF THEY PREVENTED PARA 1302 00:59:57,587 --> 00:59:58,822 APOPTOSIS KIDNEY CELLS WERE ABLE 1303 00:59:58,822 --> 01:00:00,490 TO RECOVER FAST IR, HAVE YOU 1304 01:00:00,490 --> 01:00:01,758 LOOKED AT YOUR MOUSE MODEL, IF 1305 01:00:01,758 --> 01:00:04,294 THEY ARE ABLE TO RECOVER FASTER 1306 01:00:04,294 --> 01:00:07,063 FROM LIVER INJURY, FROM -- 1307 01:00:07,063 --> 01:00:11,735 >> NO, WE REALLY HAVEN'T DONE 1308 01:00:11,735 --> 01:00:13,236 ANYTHING MORE THAN THE LIVER 1309 01:00:13,236 --> 01:00:13,703 MODELS. 1310 01:00:13,703 --> 01:00:16,539 I THINK IT'S A VERY INTERESTING 1311 01:00:16,539 --> 01:00:17,007 QUESTION. 1312 01:00:17,007 --> 01:00:18,408 WE'VE BEEN -- WE'VE GONE TO THE 1313 01:00:18,408 --> 01:00:18,942 RAT. 1314 01:00:18,942 --> 01:00:20,477 WE'VE MADE A KNOCKOUT OF NINJ1 1315 01:00:20,477 --> 01:00:24,748 IN THE RAT BECAUSE THAT'S 1316 01:00:24,748 --> 01:00:32,622 PROBABLY BETTER FOR ISCHEMIA 1317 01:00:32,622 --> 01:00:33,723 REPERFUSION. 1318 01:00:33,723 --> 01:00:34,724 THEY ARE ONGOING EXPERIMENTS. 1319 01:00:34,724 --> 01:00:36,459 I THINK THE RAT IS A BETTER 1320 01:00:36,459 --> 01:00:38,328 MODEL SO WE WENT THE EXTRA MILE 1321 01:00:38,328 --> 01:00:39,296 THERE FOR THAT. 1322 01:00:39,296 --> 01:00:43,466 >> AND THE LAST QUESTION, DOES 1323 01:00:43,466 --> 01:00:45,669 IT ALSO -- IS THIS SOMETHING YOU 1324 01:00:45,669 --> 01:00:49,272 COULD USE FOR RED BLOOD CELLS, 1325 01:00:49,272 --> 01:00:51,041 FOR DEFICIENCY, LYSIS OF RED 1326 01:00:51,041 --> 01:00:51,875 BLOOD CELLS? 1327 01:00:51,875 --> 01:00:54,177 >> I HAVE TO CONFESS I DON'T 1328 01:00:54,177 --> 01:00:55,712 KNOW. 1329 01:00:55,712 --> 01:00:58,782 YEAH. 1330 01:00:58,782 --> 01:00:59,449 >> THANK YOU. 1331 01:00:59,449 --> 01:01:00,717 >> IT'S A GOOD QUESTION. 1332 01:01:00,717 --> 01:01:07,457 YOU SHOULD COME TO GENENTECH. 1333 01:01:07,457 --> 01:01:09,526 >> WONDERFUL TALK, DR. DIXIT. 1334 01:01:09,526 --> 01:01:10,360 TWO QUESTIONS BASICALLY. 1335 01:01:10,360 --> 01:01:12,329 DO YOU THINK THAT THERE'S ANY 1336 01:01:12,329 --> 01:01:16,166 ASSOCIATION BETWEEN LIKE 1337 01:01:16,166 --> 01:01:21,404 ACTIVATION OF THE NINJ WITH 1338 01:01:21,404 --> 01:01:21,838 ACUTE INFLAMMATION? 1339 01:01:21,838 --> 01:01:22,839 >> I THINK IT'S ACUTE INJURY. 1340 01:01:22,839 --> 01:01:27,210 I THINK YOU HAVE TO OVERWHELM 1341 01:01:27,210 --> 01:01:32,449 THE EFFEROCYTOSIS MECHANISMS SO 1342 01:01:32,449 --> 01:01:37,153 DYING CELLS LICE. 1343 01:01:37,153 --> 01:01:39,255 >> ACTIVITY OF NINJ1, DO YOU 1344 01:01:39,255 --> 01:01:40,090 THINK THAT? 1345 01:01:40,090 --> 01:01:45,929 >> WELL, IN ACUTE INJURY, DYING 1346 01:01:45,929 --> 01:01:48,665 CELLS ARE NOT TAKEN UP THEY WILL 1347 01:01:48,665 --> 01:01:50,533 GO VERY RAPID NINJ1 MEDIATED 1348 01:01:50,533 --> 01:01:50,834 LYSIS. 1349 01:01:50,834 --> 01:01:53,570 I THINK THE QUESTION WE'RE 1350 01:01:53,570 --> 01:01:55,672 STRUGGLING WITH IS WHY DO YOU 1351 01:01:55,672 --> 01:01:58,074 GET FROM MONOMER TO POLYMER IN 1352 01:01:58,074 --> 01:02:00,844 DYING CELLS. 1353 01:02:00,844 --> 01:02:03,480 >> THE OTHER QUESTION, CISPLATIN 1354 01:02:03,480 --> 01:02:04,247 ASSOCIATED WITH NINJ1-ASSOCIATED 1355 01:02:04,247 --> 01:02:07,317 DEATH, SO AS WE KNOW THAT 1356 01:02:07,317 --> 01:02:10,153 CISPLATIN IS TOXIC TOWARDS THE 1357 01:02:10,153 --> 01:02:14,524 CELLS, TOXICITY IS A MAJOR CAUSE 1358 01:02:14,524 --> 01:02:15,392 FOR NINJ1 ACTIVATION OR NOT? 1359 01:02:15,392 --> 01:02:19,662 >> NO, I DON'T THINK IT WILL 1360 01:02:19,662 --> 01:02:20,663 PROTECT FROM CISPLATIN TOXICITY, 1361 01:02:20,663 --> 01:02:25,235 WE HAVEN'T DONE THOSE STUDIES 1362 01:02:25,235 --> 01:02:25,502 OURSELVES. 1363 01:02:25,502 --> 01:02:27,237 REMEMBER WITH NINJ1 THE CELLS 1364 01:02:27,237 --> 01:02:29,272 ARE DEAD. 1365 01:02:29,272 --> 01:02:29,506 RIGHT? 1366 01:02:29,506 --> 01:02:31,808 WHAT WE'RE PREVENTING IS THEIR 1367 01:02:31,808 --> 01:02:34,544 ACUTE LYSIS AND SUBSEQUENT 1368 01:02:34,544 --> 01:02:35,645 INFLAMMATORY RESPONSE. 1369 01:02:35,645 --> 01:02:35,979 >> OKAY. 1370 01:02:35,979 --> 01:02:40,550 THANK YOU. 1371 01:02:40,550 --> 01:02:43,186 >> I'LL FOLLOW UP SINCE -- YEAH, 1372 01:02:43,186 --> 01:02:49,759 I WAS WONDERING ABOUT 1373 01:02:49,759 --> 01:02:50,860 COMPENSATORY MECHANISMS IN 1374 01:02:50,860 --> 01:02:54,130 ABSENCE OF NINJ1, SURPRISED HOW 1375 01:02:54,130 --> 01:02:54,998 SOMETHING SO CONSERVED WHEN 1376 01:02:54,998 --> 01:02:56,633 DELETED IN THE MOUSE STILL 1377 01:02:56,633 --> 01:03:00,270 DOESN'T SEEM TO HAVE A MEASURE. 1378 01:03:00,270 --> 01:03:00,537 >> YEAH. 1379 01:03:00,537 --> 01:03:01,704 >> SO, YEAH -- 1380 01:03:01,704 --> 01:03:05,842 >> I THINK TWO POSSIBILITIES 1381 01:03:05,842 --> 01:03:07,610 THERE. 1382 01:03:07,610 --> 01:03:10,647 ONE IS THAT NORMALLY DYING CELLS 1383 01:03:10,647 --> 01:03:13,116 ARE TAKEN UP BY EFFEROCYTOSIS, 1384 01:03:13,116 --> 01:03:15,485 THERE'S NO DEFECT IN THE ABSENCE 1385 01:03:15,485 --> 01:03:17,554 OF NINJ1 SO IT'S ONLY WHEN YOU 1386 01:03:17,554 --> 01:03:18,655 STRESS THE MODEL WITH 1387 01:03:18,655 --> 01:03:23,793 OVERWHELMING INJURY YOU SEE A 1388 01:03:23,793 --> 01:03:24,194 PHENOTYPE. 1389 01:03:24,194 --> 01:03:25,628 THE OTHER POSSIBILITY, RELATED, 1390 01:03:25,628 --> 01:03:29,599 I DIDN'T GO INTO NINJ2 IN 1391 01:03:29,599 --> 01:03:29,833 MAMMALS. 1392 01:03:29,833 --> 01:03:31,301 SO THAT'S A WHOLE BAG OF WORMS 1393 01:03:31,301 --> 01:03:35,171 THAT I DIDN'T WANT TO OPEN UP. 1394 01:03:35,171 --> 01:03:37,574 SO WHAT THAT'S DOING, WHETHER 1395 01:03:37,574 --> 01:03:39,309 THERE'S REDUNDANCY WITH THAT, IS 1396 01:03:39,309 --> 01:03:44,581 A POSSIBILITY. 1397 01:03:44,581 --> 01:03:48,418 BUT I PERSONALLY FAVOR THAT 1398 01:03:48,418 --> 01:03:50,153 EFFEROCYTOSIS MECHANISMS ARE SO 1399 01:03:50,153 --> 01:03:53,690 EFFICIENT UNLESS YOU HAVE 1400 01:03:53,690 --> 01:03:54,557 OVERWHELMING INJURY, DYING CELLS 1401 01:03:54,557 --> 01:03:56,459 ACCUMULATE YOU'RE NOT GOING TO 1402 01:03:56,459 --> 01:03:59,929 SEE A NINJ1 PHENOTYPE. 1403 01:03:59,929 --> 01:04:04,000 >> I GUESS DURING LIKE THE AGING 1404 01:04:04,000 --> 01:04:05,068 PROCESS, THESE PROCESSES, WOULD 1405 01:04:05,068 --> 01:04:05,802 ACCUMULATE. 1406 01:04:05,802 --> 01:04:06,503 >> YEAH. 1407 01:04:06,503 --> 01:04:08,171 >> SO WHETHER THE AGED -- YOU 1408 01:04:08,171 --> 01:04:11,975 KNOW, WHETHER AGED MICE BEGIN TO 1409 01:04:11,975 --> 01:04:14,043 SEE GREAT -- YOU KNOW, THERE'S 1410 01:04:14,043 --> 01:04:16,379 GOING TO BE MORE DAMAGE. 1411 01:04:16,379 --> 01:04:18,014 >> YEAH, NO, IT'S A GOOD -- I 1412 01:04:18,014 --> 01:04:19,549 THINK WE'RE AGING A COHORT OF 1413 01:04:19,549 --> 01:04:27,123 MICE SO WE'LL SEE. 1414 01:04:27,123 --> 01:04:27,991 >> OKAY. 1415 01:04:27,991 --> 01:04:28,358 ONE MORE. 1416 01:04:28,358 --> 01:04:31,060 SO, HAVE YOU HAD THE CHANCE TO 1417 01:04:31,060 --> 01:04:31,928 LOOK FOR EXAMPLE AT ALZHEIMER'S 1418 01:04:31,928 --> 01:04:35,098 DISEASE WHERE YOU HAVE LIKE 1419 01:04:35,098 --> 01:04:35,999 ACCUMULATION OF PHAGOCYTIC 1420 01:04:35,999 --> 01:04:37,867 CELLS, HAVE A MAJOR ROLE, AND 1421 01:04:37,867 --> 01:04:39,836 THEY WOULD BE SOMEHOW 1422 01:04:39,836 --> 01:04:42,238 OVERWHELMED, OR DO YOU SEE SOME 1423 01:04:42,238 --> 01:04:44,307 CELLS BEING MORE RESISTANT TO 1424 01:04:44,307 --> 01:04:45,241 THIS MECHANISM? 1425 01:04:45,241 --> 01:04:45,875 >> TO THE NINJ1? 1426 01:04:45,875 --> 01:04:46,509 >> UH-HUH. 1427 01:04:46,509 --> 01:04:47,744 >> NO, NO, WE HAVEN'T LOOKED. 1428 01:04:47,744 --> 01:04:48,912 YOU KNOW, THIS IS WONDERFUL. 1429 01:04:48,912 --> 01:04:50,146 THIS IS A NEW DISCOVERY. 1430 01:04:50,146 --> 01:04:52,248 THIS IS WHY I ENJOY SCIENCE. 1431 01:04:52,248 --> 01:04:53,650 IT'S LIKE NEW NINJ1, RIGHT? 1432 01:04:53,650 --> 01:04:58,188 IT'S NOT LIKE I'M STUDYING MYC. 1433 01:04:58,188 --> 01:04:58,655 [LAUGHTER] 1434 01:04:58,655 --> 01:04:59,822 YOU KNOW. 1435 01:04:59,822 --> 01:05:00,690 IT'S NEW! 1436 01:05:00,690 --> 01:05:01,324 I DON'T KNOW! 1437 01:05:01,324 --> 01:05:03,993 I WILL MAKE THE REAGENTS 1438 01:05:03,993 --> 01:05:04,360 AVAILABLE. 1439 01:05:04,360 --> 01:05:06,930 EVERYBODY SHOULD -- JUST LIKE 1440 01:05:06,930 --> 01:05:07,797 WITH GASDERMIN-D IN 2015, IT 1441 01:05:07,797 --> 01:05:07,964 WAS. 1442 01:05:07,964 --> 01:05:09,666 NEW WE MADE THE REAGENTS 1443 01:05:09,666 --> 01:05:10,200 AVAILABLE. 1444 01:05:10,200 --> 01:05:10,767 IT'S NEW! 1445 01:05:10,767 --> 01:05:11,201 I DON'T KNOW. 1446 01:05:11,201 --> 01:05:12,869 THERE ARE HUNDREDS OF QUESTIONS 1447 01:05:12,869 --> 01:05:15,038 YOU COULD ASK. 1448 01:05:15,038 --> 01:05:16,039 MORE PEOPLE, BETTER. 1449 01:05:16,039 --> 01:05:19,642 AND REMEMBER WE MAKE REAGENTS 1450 01:05:19,642 --> 01:05:20,076 AVAILABLE. 1451 01:05:20,076 --> 01:05:23,379 >> I THINK I'M GOING TO ASK ALSO 1452 01:05:23,379 --> 01:05:27,183 A QUESTION THAT YOU MAY NOT 1453 01:05:27,183 --> 01:05:27,617 KNOW. 1454 01:05:27,617 --> 01:05:31,554 BUT, YOU KNOW, YOU HAVE BEEN 1455 01:05:31,554 --> 01:05:35,825 SAYING THAT INHIBITING NINJ1 1456 01:05:35,825 --> 01:05:36,626 INHIBITS STERILE INFLAMMATION 1457 01:05:36,626 --> 01:05:42,932 BUT THE SAME TIME YOU SHOWED US 1458 01:05:42,932 --> 01:05:44,267 ACTUALLY GASDERMIN IS THERE AND 1459 01:05:44,267 --> 01:05:46,803 RELEASING EVERYTHING THAT YOU'VE 1460 01:05:46,803 --> 01:05:47,070 MEASURED. 1461 01:05:47,070 --> 01:05:47,770 RIGHT? 1462 01:05:47,770 --> 01:05:48,605 >> YES. 1463 01:05:48,605 --> 01:05:49,138 >> SO -- 1464 01:05:49,138 --> 01:05:50,773 >> YES, SO THE GASDERMIN 1465 01:05:50,773 --> 01:05:54,477 REMEMBER HAS A MOLECULAR WEIGHT 1466 01:05:54,477 --> 01:05:54,811 CUTOFF. 1467 01:05:54,811 --> 01:05:59,082 >> YEAH, SO BASICALLY YOU'RE 1468 01:05:59,082 --> 01:06:00,250 SAYING THAT THIS, YOU KNOW, AT 1469 01:06:00,250 --> 01:06:05,555 THE SAME TIME AT LEAST THE DATA 1470 01:06:05,555 --> 01:06:09,525 THAT YOU HAVE SHOWN DON'T 1471 01:06:09,525 --> 01:06:10,660 REALLY -- SO YOU'RE SAYING THAT, 1472 01:06:10,660 --> 01:06:13,496 YES, THERE ARE THESE LARGER 1473 01:06:13,496 --> 01:06:16,899 MOLECULES THAT MUST BE RELEASED 1474 01:06:16,899 --> 01:06:18,768 FOR THE ENTIRE INFLAMMATORY 1475 01:06:18,768 --> 01:06:21,638 CASCADE TO REALLY HAPPEN, RIGHT? 1476 01:06:21,638 --> 01:06:22,939 >> YEAH, YOU KNOW, I THINK YOU 1477 01:06:22,939 --> 01:06:27,443 MAKE A VERY GOOD POINT. 1478 01:06:27,443 --> 01:06:31,581 I THINK THAT WITH THE 1479 01:06:31,581 --> 01:06:32,448 GASDERMIN-D INJURY, I NEVER 1480 01:06:32,448 --> 01:06:37,186 THOUGHT I WOULD SAY THAT BUT I 1481 01:06:37,186 --> 01:06:39,022 THINK THAT IS POTENTIALLY 1482 01:06:39,022 --> 01:06:39,689 REVERSIBLE. 1483 01:06:39,689 --> 01:06:42,191 SO THERE'S SOME DATA IN THE 1484 01:06:42,191 --> 01:06:45,495 LITERATURE, AND OUR OWN DATA, WE 1485 01:06:45,495 --> 01:06:52,802 MADE HUNDREDS OF ANTIBODIES TO 1486 01:06:52,802 --> 01:06:54,203 GASDERMIN-D, MANY THOUSANDS, 1487 01:06:54,203 --> 01:06:55,972 SHEEP-LOOKING CREATURES, LLAMAS 1488 01:06:55,972 --> 01:06:57,040 OR SOMETHING. 1489 01:06:57,040 --> 01:07:01,444 AND THEN THE TECHNICIAN CAME IN 1490 01:07:01,444 --> 01:07:05,181 MY LAB, FAIRLY SENIOR SCIENTIST, 1491 01:07:05,181 --> 01:07:06,616 I WONDER IF ANY INHIBIT 1492 01:07:06,616 --> 01:07:07,950 GASDERMIN-D FUNCTION? 1493 01:07:07,950 --> 01:07:11,521 THAT'S A REALLY, REALLY BAD 1494 01:07:11,521 --> 01:07:13,723 IDEA, BECAUSE GASDERMIN-D'S 1495 01:07:13,723 --> 01:07:15,692 INSIDE THE CELLS, AND IT'S 1496 01:07:15,692 --> 01:07:17,560 GETTING ACTIVATED. 1497 01:07:17,560 --> 01:07:18,761 YOU'RE ADDING THE ANTIBODY 1498 01:07:18,761 --> 01:07:21,764 OUTSIDE THE CELLS, AND WE HAVE A 1499 01:07:21,764 --> 01:07:22,165 MEMBRANE. 1500 01:07:22,165 --> 01:07:24,701 NOT GOING TO WORK. 1501 01:07:24,701 --> 01:07:25,835 DON'T DO EXPERIMENT. 1502 01:07:25,835 --> 01:07:28,338 SHE DID IT ANYWAY. 1503 01:07:28,338 --> 01:07:28,871 [ LAUGHTER ] 1504 01:07:28,871 --> 01:07:31,174 AND THERE IS A NANOBODY, SERIES 1505 01:07:31,174 --> 01:07:32,475 OF NANOBODIES THAT WE'VE 1506 01:07:32,475 --> 01:07:34,977 DISCOVERED BUT ALSO BEEN 1507 01:07:34,977 --> 01:07:37,180 REPORTED BY OTHERS, THAT WILL 1508 01:07:37,180 --> 01:07:38,381 ACTUALLY REVERSE THAT INJURY. 1509 01:07:38,381 --> 01:07:42,318 SO THERE IS A CAPACITY. 1510 01:07:42,318 --> 01:07:44,053 SO WHAT THAT SAYS, FOR MEMBRANE 1511 01:07:44,053 --> 01:07:45,722 DAMAGE YOU HAVE TO EXCEED A 1512 01:07:45,722 --> 01:07:49,325 THRESHOLD FOR IT TO BECOME 1513 01:07:49,325 --> 01:07:49,792 IRREVERSIBLE, RIGHT? 1514 01:07:49,792 --> 01:07:52,695 I DON'T THINK THERE'S ANY GOING 1515 01:07:52,695 --> 01:07:55,665 BACK FROM NINJ1 THOUGH. 1516 01:07:55,665 --> 01:07:58,735 ONCE THAT'S ACTIVATED, KABOOM. 1517 01:07:58,735 --> 01:08:01,404 >> THANK YOU. 1518 01:08:01,404 --> 01:08:04,307 >> A PHILOSOPHICAL -- 1519 01:08:04,307 --> 01:08:07,176 >> THANK YOU FOR THE TALK. 1520 01:08:07,176 --> 01:08:10,513 MAYBE THE LAST TWO. 1521 01:08:10,513 --> 01:08:17,420 ACTUALLY VERY BEGINNING OF THE 1522 01:08:17,420 --> 01:08:20,256 NINJ1 FINDINGS, IN ACTIVATION, 1523 01:08:20,256 --> 01:08:20,723 REQUIRED. 1524 01:08:20,723 --> 01:08:25,294 AND THEN SO SEEMS LIKE THERE'S 1525 01:08:25,294 --> 01:08:32,969 WHY ACTIVATED CANNOT BE SITUATED 1526 01:08:32,969 --> 01:08:34,137 THROUGH THE NINJ1 1 1527 01:08:34,137 --> 01:08:35,471 (INDISCERNIBLE). 1528 01:08:35,471 --> 01:08:39,642 >> YEAH, I WOULD SAY THAT IF YOU 1529 01:08:39,642 --> 01:08:42,044 DON'T HAVE GASDERMIN-D, YOU'RE 1530 01:08:42,044 --> 01:08:46,182 NOT GOING TO RELEASE IL-1 BETA. 1531 01:08:46,182 --> 01:08:48,518 BUT EVENTUALLY THE CELL WILL 1532 01:08:48,518 --> 01:08:50,153 LYSE AND YOU'LL RELEASE IL-1 1533 01:08:50,153 --> 01:08:50,987 BETA. 1534 01:08:50,987 --> 01:08:52,889 SINCE YOU'VE NOT ACTIVATED OR IF 1535 01:08:52,889 --> 01:08:54,323 YOU HAVEN'T ACTIVATED THE 1536 01:08:54,323 --> 01:08:56,826 CASPASES, IT WILL BE UNPROCESSED 1537 01:08:56,826 --> 01:09:00,863 IL-1 BETA. 1538 01:09:00,863 --> 01:09:04,934 IF YOU'VE ACTIVATED CASPASES 1539 01:09:04,934 --> 01:09:08,404 YOU'LL RELEASE WITH GREATLY 1540 01:09:08,404 --> 01:09:08,905 DELAYED PAN KINETICS, 1541 01:09:08,905 --> 01:09:10,706 INDEPENDENT MANNER, BUT I DON'T 1542 01:09:10,706 --> 01:09:11,407 THINK THAT'S PHYSIOLOGICALLY 1543 01:09:11,407 --> 01:09:14,844 RELEVANT BECAUSE OF ALL THE 1544 01:09:14,844 --> 01:09:16,078 STUDIES WE'VE DONE IN VIVO 1545 01:09:16,078 --> 01:09:17,513 CHALLENGES. 1546 01:09:17,513 --> 01:09:22,318 SO AS YOU SAW THAT WE'RE ABLE TO 1547 01:09:22,318 --> 01:09:24,053 COMPLETELY SUPPRESS IL-1 BETA 1548 01:09:24,053 --> 01:09:25,188 RELEASE IN A GASDERMIN-D 1549 01:09:25,188 --> 01:09:27,623 KNOCKOUT. 1550 01:09:27,623 --> 01:09:27,890 >> OKAY. 1551 01:09:27,890 --> 01:09:30,059 THANK YOU. 1552 01:09:30,059 --> 01:09:33,529 1553 01:09:33,529 --> 01:09:35,097 [APPLAUSE] 1554 01:09:35,097 --> 01:09:36,098 1555 01:09:36,098 --> 01:09:38,601 [END OF PROGRAM] 1556 01:09:38,601 --> 01:09:46,375