Batteries  one  of  these  Testing. So,  make  sure  it's  picking  the  body  here. Testing,  one,  two,  three,  there. Three  batteries  should  be  okay? Yeah,  yeah,  should  be.  Let's  take this  out  of  you.  Should  good. Okay. Let's Batteries  in  this  too? Do  you  think  you  would  want  this  monitor  on? I  didn't  ask  him  about  it. I  didn't  ask.  Okay.  I  can. Yes. Okay.  Is  recording  Yes  recording. Do  you  want  to  have  the  chat? Turn  on  because  if you  turn  it  on  now,  we  can  close  the  window, but  if  someone  turns it  on  a  Stop letting  me  sign  on  in  two  places. A, What  would  you  like  for  lunch? If  you  want. They're  all sandwich  have one  wrap  water? Yeah. Okay. Oh,  right. I  had  a  chance  you  want  to  get the.Exce  so  we  have this  monitor  here  that  kind  of shows  you  your  notes  and everything  and  also  the  next  slide. Okay.  Do  you  want  to  turn  it  off  Okay. I  switch  off. Sometimes  we  don't  turn  it  on,  but  we  can. Yeah,  it's  nice.  All  right, you  have  everything  you  good  luck. Er.  Thank  you. Good?  Okay.  I  didn't  see  it  doesn't  do  that  anymore. There. I  like  to do  that  that  one  this  is. I  can't  log  on  in  two  places  anymore. So  I  have  to  use  a  different  email  address  to  log. Okay. Okay. But  I need  to  be  able  to  chat  with. Okay.  I  should  just  set  that  up. Yeah, I  should add start to  take  this  over. Oh,  but  you  know  you  can  tell  him  not  to. So  when  you  join,  just  say  you  want  to  switch  justTanks Microphone  for  that  instruction.  I  don't  have  to. Okay. Yeah.  Yeah,  part  of  that. So  one  of  the  questions  came  up  with  it? Does  that  mean  to So Yeah,  I'll  highlight.  Yeah. Yeah, I awesome  to  share  back. Yeah. No,  they have  yeah. 30  years  ago. Hi,  I'm  Thank  you. In. I  give  the  grand. During  COVID  it's popular  now,  I  think. Kind of  freaking  out. I  was  like,  I  kind  of expected  yeah,  I'm  glad  I  said. Although  I  sent  west. Your  advisor was  two  No, no,  so  if  I'm  adding  Sam  as  my, by  two  more  only two  con  Simon  yeah. Yeah.  I'm  going  to ask  and  what  that  might  look  like. 2001,  they  did  there. Yeah,  yeah.  I  think  that  should  have  ended. I  should  Yeah. Yeah.  Yeah,  that's  what  I  was  talking about  dedicated  to  learning. Yeah. So you  have the  interest  as  far  as  sellex  systems  or behavior.  Yeah,  I  think. So go. I'm  glad  you  made  it.  Okay. Okay. Yeah. I so  I've  been able  to  enter  by  the  signs. I  was  like,  myself, we're  going  to  be but Oh. I  hope  you  all  but  this  is  so  weak. No  like  this  only  happened  for  the  for  110  years. So  my  family  really  enjoyed  it. I  hope  you  get  more  cv. So  it's  really  a  great  honor  to have  doctor  Sim  Jung  down  here  with  us  today. Doctor  Dong  earned  his  bachelor  degree  in  chemistry  from Holy  Crowd  College  and  went  on  to  complete his  PhD  with  doctor  Larry  Diversity  at  UCLA. So  doctor  Dong  his  post  of training  with  doctor  David  Anderson  at  Cal  high. Um,  Bob  John  currently  is now  a  professor  in  neuroscience, neurosurgery  and  dermatology  at  Johns  Hopkins  University. So  he  received  HHMI  young  Investigator  of  2009  and  became an  HHII  investigator  in  Anti  psychology. His  research  has  identified  a  family  of de  protein  coupled  receptors that  regulate  pain  and  each  sensation, which  has  led  to  the  development  of normal  tech  compound  clinical  time. It  also.  Almost  every coastal  like  I  start  and  I  will  latch I'm  looking  forward  to  hear  about  this  first  I  want  to pen  Thank  you  very much  for  the  nice  introduction and  thank  you  for  coming  to  my  talk, and  it's  always  the  best  feeling  to  see the  former  Tinse  achieve  great  success  like  Leon. And  so  happy  New  Year,  everyone. And  so  today,  I'm  going  to  talk  about  this  GPCR  family. I  cloned  when  I  was  a  poster  and David  Anderson  left  and  sorry. Down.  Freeze  here. Yeah,  I  think  you  put  in  there. Volunteer.  Yeah. Okay,  maybe  I  just  do  manually  then. Okay,  now  it  works. Yeah.  Okay.  Great.  So  sorry  for  the  glitches. And  so  the  RG  include  large  family  DPCR  and  there are  50  mouth  Rgs  shown  here  in  family  and  18  human  Rgs. And  the  interesting  thing  about  this  family  is  on evolved  very  recently  is  only  evolved  in  the  tetrapod, the  animal  with  four  limbs. Now  it  makes  sense  because  only  the  animal with  four  limbs  can  scratch  their  edge. And  so  they  don't  exist  in  my  fish,  actually. So  here,  most  of  the  MRGs  are clustered  on  the  mouse chromosome  seven,  human  chromosome  11. I  show  the  gene  cluster  here and  each  bar  represent  one  RG. And  the  RG  will  first  identify  in sensory  neuron  and  later  on, found  in  other  immune  cells  like mass  cell,  neutrophil  and  basophil. And  based  on  the  sequence, based  on  the  expression  pattern and  also  the  vegan  specificity, we  can  assign  different  human  homolog  shown  here. So  this  gene  family,  as  shown  here, expressing  diverse  cell  types, and  I  have  a  different  biological  functions. So  let  me  tell  you  the  story  on  this  receptor as  each  receptor  first.  So  what  is  H? H  is  defined  by  this  German  physician more  than  360  years  ago as  unpleasant  sensation  to  make  you  want  to  scratch. And  although  it's  a  very  simple  definition, but  it's  still  accurate, so  we  currently  still  use  it. But  why  do  we  need  H? So  H  is  considered as  a  protective  mechanism  just  like  a  pain, and  you  want  to scratch  to  remove  irritant  from  the  skins. And  other  people  think  scratching  can  actually mobilize  immune  cells  in  the  skin, so  you  have  more  immune  response, and  also  it  can  call  our  attention  to the  affected  skin  areas  and  avoid  irritant  in  the  future. And  also  the  scratching can  really  provide  temporary  relief, so  you  feel  good,  so  that's  why  you're  scratching. However,  the  pathological  chronic  it  can lead  distress  and  suffering  doesn't have  much  beneficial  value  for  chronic  itch. So  the  work  from  Mylab  and  many  other  lab  now  show that  each  is  a  distinct  sensation  from  pain  sensation. And  also  the  beautiful  work  from Leons  while  she  was  a  poster  in  MLf and  showed  there's  each  specific  neuron in  dorsal  root  ganglions. And,  we  also  find  the  MRGs  the  receptor  with  clone. Some  of  them  actually  expressing dorsal  root  galleon  sensory  neuron, and  these  are  the  neurons  and  axon  to  the  skin. And  some  of  these  RG  is  function  as a  novel  H  receptor  mediating  non  chimergic  age, just  give  you  an  example. So  this  receptor  is  called  MRGD. It's  a  receptor  for  beta  onin. So  if  you  do  body  building, you  probably  heard  of  beta  onin because  the  body  builder  tend  to  take  a  lot of  uh  beta  one  and  other  supplement  to  build  muscles. And,  however,  they  all  experience very  strong  itch  as  a  side  effect. It  turned  out  this  beta  on combines  MRDD  to  generate  this  irritating  itch  sensation. And  besides  those  acute  itch, when  the  itch  lasts  longer  than  like  four  or  five  weeks, it  becomes  chronic,  and  that's  a  disease  state. And  as  you  know,  many  skin  disease  like acma  and  psoriasis  or  different  type  of dermatitis  can  also  cause  chronic  itch. And  then  the  systemic  disease like  liver  disease  or  kidney  disease can  also  accumulate  a  lot  of metabolite  in  the  system  to  cause  chronic  itch. And  drugs  induce  side  effects, each  side  effect  is  also  a  big  problem  like morphine  ng  morphine  can  cause  a  lot  of  itch. Uh,  also,  there's a  neurological  neuropathic  age  like  shingles and  so  then  this  over  2000  years  ago, the  Greek  physician  made  a  striking  notice, and  they  found  a  lot  of yellow  skinned  people  tend  to  complain  intensive  itch. Now  we  know  this  condition  is  called  jaundice. So  the  jaundice  is  actually  caused  by  accumulation of  the  yellow  metabolite  called  Blrubin  in  the  system. And  the  Blrubin  is  a  degradation  product  from  him. And  normally  it  can  excrete  it  in  the  urine  and  feces. And,  however,  if  you  have a  liver  damage  and  this  secretion  passes  block, the  bilirubin  can  accumulate  a  very  high  level  to cause  yellow  skin  and  also  cause  itch  sensation. And  so  basically  the  jaundice  associate  itch commonly  occurred  in  the  context  of  liver  damage, like  clostsis  like  the  blockage and  stoppage  of  the  bile  flows. And  so  this  costsis  can  cause  by many  different  liver  injuries. And  many  people  in  the  US actually  suffer  from  this  condition. However,  currently,  there's no  effective  treatment  for  closty  itch. So  when  a  talented  former  MD  PhD  student Jimmy  entered  the  lab, he  wanted  to  know  why  or  how  Billy  Rubin  can  cause  it. So  First  of  all, I  just  tell  you  how  do  we  study  itch  in  mice, and  it  turned  out  very  simple. We  just  inject  whatever  itchy chemicals  into  the  back  of  the  skin  in  the  neck, in  the  mice  and  like  his  tomy  or  Bilirubin, you  can  watch  them  for  record  them  for  half  hour, as  you  can  see  here,  the  mice  actually  can  elicit this  characteristic  scratching  behavior using  the  hind  paw  and  scratch at  high  frequency,  about  ten  hertz. So  you  can  just  videotape  for  30  minutes and  count  how  many  times  they  scratch  and  quantify  that. And,  so  Jimmy  actually  did  the  experiment, so  he  inject  Blrubinto  the  mole  skin  and  increase  dose, you  can  see  here,  Bilirrubin  can  induce dose  dependent  scratching  behavior in  Wild  time  mice  shown  here. And  he  also  pre  treated  the  mice  with  antihiamin. However,  the  antihamine  did  not reduce  Billy  Rubin  induced  age. So  suggesting  that  Bilirubin  cause  non  homergic  itch. And  then  he  wanted  to  know  whether  there  is a  bilirubin  receptor  existing  mouse  genome. So  he  actually  screened  all  the  mouse  Rgs  and  human  Rgs against  bilirubin  and  using the  standard  calcin  imaging  as  a  read  off  for  GPCRs. As  you  can  see  here,  he  found mouse  MRG  A  one  and  human  genes  called  MRGX four  and  show  a  pretty  nice  dose  response  curve In  response  to  a  Bilirubin  stimulation  consistently. And  we  also  did  a  binding  essay, and  we  found  Bilirubin  can  directly  binds  to these  two  receptors  at the  EC  50  around  the  micromolar  range. So  this  is  the  range  you  can find  in  the  colostic  patients. And  it  turned  out  most  of  the  MRGs  are  low  affinity. So  the  micromolar  is 50  or  low  affinity  and  broadly  tuned  receptor. So  later  on,  three  D  structure  actually  can show  why  this  receptor are  low  affinity  and  broadly  tuned. So  then  Jimmy  Fund, this  receptor  is  also  specifically  expressed  in  a  subset of  DRG  neurons  as  shown  as  a  green  staining  here. And  then  he  also  tried  to  see  whether  Bilirubin  can really  excite  the  neuron  by  using  patch  claim  recording. As  you  can  see  here,  Bilirubin  can induce  very  robust  action  potential in  the  wildtype  DRG  neuron. However,  if  you  apply  Bilirubin  into  the  MGA  one, knockout  DRG  neuron,  there's  no  activation  at  all. And  consistently,  if  you  just  inject the  BDI  Rubin  into  the  MGA  one  knockout  new  mice, you  see  the  BDI  rubin this  scratching  are  significantly  reduced. So  suggesting  the  MGA  one  mediate  BDI  rubind  edge. So  those  are  the  injecting  the  exogenous  bilirubin. Here,  Jimmy  actually  developed a  colocated  model  for  mice. So  he  actually  used  a  chemical  to  damage  the  liver  to block  the  excretion  of  Brubin. So  as  you  can  see  here, after  those  liver  damage  or  this  colossus  condition, there  is  a  significant  increase  in the  bilirubin  level  in the  mice  in  both  wild  type  and  MRG  knockout  mice. However,  these  mice  actually can  develop  a  spontaneous  scratching because  accumulation  of  Dlubin  presumably. You  can  see  the  Wilt  mice  after  the  liver  damage, they  scratch  significantly  higher  than  the  control  mice. And  however,  the  MRGA one  mice  scratch  much  lower  than  the  Wilt  mice. Again,  this  data  suggests  the  MRGA  one actually  mediate  lostatic  itch. And  then  we  collaborate  with  physicians  actually  collect the  serum  from  several  lostic  patients. So  this  patient  actually  suffered  very  severe  itch and  their  bilirubin  level  are significantly  increased  as  we  measured. And  then  Jimmy  actually  can  inject this  serum  either  from  the  healthy  controls or  from  six  costic  patient  individuals into  the  wildtype  and  also  MRGA  one  narco  mice. And  the  healthy  control  serum  did  not  induce significant  scratching  between  wildtype  and  narco  mice. However,  for  Closytic  patient  serum, they  induce  a  significant  higher  scratching compared  to  the  scratching  in  the  MRGAONaco  mice. So  human  phytic  serum  can  cause  itch  in  mice. So  then  we  want  to  know  what's  in the  serum  really  cause  this  itch,  right? Is  Brubin  really  the  factor? So  Jimmy  actually,  uh, using  two  different  methods  to deplete  or  block  the  Bilirubin. First  of  all,  he  used iron  chloride  to  deplete  Bilirubin  from  the  serum. Another  approach,  he  used  Bilirubin  antibody. In  both  cases,  you  can  see  at  the  green  bar, the  scratching  are  significantly  reduced. So  if  you  remove  Bilirubin  from  the  serum, you  can  have  much  less scratching  suggesting  that  Bilirubin  is a  major  contributor  in  the  serum  to  cause  itch. So  here,  later  on, Jimmy  and  several  other  lab  showed the  human  version  of  this  receptor not  only  binds  to  bilirubin, but  it  can  also  activate  another  lipid  called  bio  acid. So  bio  acid  has  shown  to  be a  major  factor  for  the  costed  edge. Now  we  identify a  novel  bio  acid  receptor  mediate  bio  acid induced  costed  g.  So just  summarize  this  part  of  the  work. So  we  found  the  mouse  RG  A  one, human  MgXour  a  bilirubin  receptor, and  human  RG  X  four  is  a  bio  acid  receptor, and  they  all  mediate  closeted  edge. And  so  now  I want  to  switch  the  gear  a  little  bit to  talk  about  other  Rgs. We  found  some  other  Rgs  not  expressed  in  sensory  neuron, but  rather  express  in innate  immune  cells  like  mass  cells, neutral  fuel  and  basal  fuels. And  so  today  I  will  tell  you  several  story about  mass  cell  receptors  and  also  neutro  field  receptor. And  they  play  an  important  role  in plain  H  and  also  host  defense. So  what  is  cells? So  us  cell  are  the  first  responder  cells upon  infection  injury. So  they  are  the  tissue  resident  cells. You  can  find  them  in  the  skin  in  many  other  tissues. And  so  the  major  characteristic of  this  type  of  cells  are  they  can  when  they  activate, they  can  degranulate  and  release the  famous  histamine  to  cause  allergic  reactions. And  they  also  can  release  serotonin  proteins  and cytokines  and  to  recruit other  immune  cells  and  promote  inflammation. And  so  there's  two  major  pathway to  active  ma  cell  and  degraded  mas  cell. One  major  pathway  is  called  IGE  and  FC  receptors. So  this  is  antibody  IGE  and  you  can have  antigen  specific  IG  bind  the  antigen  and  cross link  the  the  FC  receptor  to induce  cell  degranulation  to cause  classic  allergic  reactions. And  then  there's  another  major  pathway people  notice  for  decades, and  that  is  the  pathway  activated  by the  so  called  pose  charge  substance collectively  called  basic  secreticGag. So  basic  means  postechart secret  gas  means  make  cell  to  secrete. And  there's  many  positive  chart  peptides  and compounds  can  activate  my  cell  to  degranulate. And  however,  the  receptor  is unknown  for  many  years  until  our  discovery. So  former  poster  Benjamin  McNeil actually  identified  these  basic  security  guards. And  so  he  found  this MRG  B  two  is  exclusively  expressed  in  mouth  cells. And  so  we  generate  MRGB  two  cre  lines  and  use did  metal  to  label  the  MRGB  two  expressing  cells. And  we  found  100%  of  this  receptor expressed  in  cells  and shown  using  another  marker  called  evidence. So  there's  100%  overlap, and  you  can  find  these  positive  cells  in  many  tissue, including  skins,  airway  and  heart. And  and  this  is also  consistent  with  online  gene  chip  data. So  people  find  by  sequencing  that  mouse  MRGB two  are  exclusively  expressing my  cells  and  not  in  any  other  tissue, not  in  DRG  either. And  later  on,  people  find  the  human  homologs  called RG  X  two  is  also  specific  expressing  human  cells. So  to  make  a  long  story  short, and  Ban  actually  use  multiple  approaches  and  find these  basic  secudigag  shown here  can  specifically  activate  these  two  receptors. For  example,  compound  48 is  most  commonly  used  secreto  gag. Degranded  cell,  it  can  activate  both  receptors, and  the  substance  P  is  a  famous  neuropeptides. It's  also  positive  charge, can  also  activate  these  receptors. And  besides  the  basic  security  guards, and  Ben  also  found many  other  drugs  can  cause anaphylatic  reactions  shown  here, can  also  activate  these  receptors,  okay? And  so  these  drugs  are  commonly  used  in the  clinic  to  treat  different  kind  of  conditions. And  so  what  is  anaphylatic  shock? So  this  is  a  relatively  common  conditions, especially  during  surgeries, so  it  can  be  life  threatening, so  you  can  have  a  very  sudden  drop  in the  body  temperature  and lower  blood  pressure  and  short  of  brightness, breath  and  can  cause  by  food,  venom  and  medications. So  it's  pretty  serious  condition, and  to  our  surprise, all  those  drugs  cause  anaphylatic  reactions can  robustly  activate  wild  type  mus  cells, and  nearly  100%  all  the  muscle  responds  to  those  drugs. However,  in  MRGB  two  knockout  muscle, they  don't  respond  at  all. It's  very  clean  result. And  you  can  actually  induce anophlatic  reaction  by  tail  away  injection, one  of  the  antibiotic  called  separate  flaxation. So  if  you  inject  tail  away inject  hydros  a  very  quick  shorter  time, you  can  actually  see  the  anophalatic  reactions. So  here  just  show  the  body  temperature  is dramatically  decrease within  a  few  minutes  in  the  wilt  mice. And  many  of  these  mice  actually  died  because  of  shock. And  then  if  you  look  at  the  knockout,  they  are  fine, so  none  of  them  actually killed  by  these  anaphylatic  reactions. So  this  data  indicate this  muscle  specific  receptor  MIGB  two, a  mediate  drug  induced  anaphylatic  reactions. So  why  this  receptor  can  have so  many  ligands  and  activism? So  Brian  Ross's  lab  at UNC  and  another  lab  in  China,  actually, they  solve  a  three  D  structure  of this  human  receptor  MRX  two  using  Crow  EM. And  they  found  this  binding  pet, this  receptor  has  very  shallow, so  it  can  fit  a  lot  of  ligand  into  it. So  the  shallow  bending  pack fed  the  ideas  from  misc  receptor, and  they  also  find negative  charged  residue  actually can  interact  with  the  positive  charged  ligands. So  basically,  their  three  D  structure consolidate  with  our  biology  data  pretty  well. So  then  we  also  want  to  know  whether this  muscle  receptor  play  a  role  in  pain  sensation. So  former  host  Dustin  Green want  to  know  whether  this  receptor, the  muscle  receptor,  have  a  role  in  pain  or  not. So  as  you  know, um  substance  P  is a  key  neuropeptide  mediate a  process  called  neurogenic  inflammation. So  when  you  have  a  tissue  injury, the  injured  nerve  actually  can  secrete neuropeptides  like  substance  P.  And  then  substance  P can  activate  nearby  muscells  to  induce  inflammation  by secreting  cytokine  chemokines  to recrut  other  immune  cells. So  this  process  called neurogenic  inflammation  because  the  inflammation is  caused  by  nerve  injuries. Through  the  substance  P  release. So  the  dogma  field  in  the  pain  field suggests  there's  a  canonical  receptor or  conventional  receptor  for substance  called  pachycinRceptor  or K  one  receptor  function  as a  substance  P  receptor  mediate  neurogenic  inflammation. However,  the  antagonist, the  clinical  trial  for  the  antagonist  NK  one failed  with  a  very  disappointed  outcome because  they  don't  have  any  energetic  effect. So  what  is  going  on  here? Dustin  just  perform  very  simple  experiment. You  want  to  see  whether  the  K  receptor is  really  expressing  muscle  or  not. As  you  can  see  here,  the  N  by  using RTPCR  he  only  detect  NK  one  receptor, the  carmonic  receptor  of  substance  P, only  in  the  spinal  cord, but  not  in  the  DRG  sensory  neuron. And  more  importantly,  it's  not  in  mass  cells. On  the  other  hand,  our  receptor MGB  two  are  highly  expressed  cells, not  in  DRG,  not  in  the  spinal  cord. So  it  is  MRGB  two, but  not  K  one  expressing  cells. So  consistently,  when  we  apply  substance  P  onto  the  cell, as  you  can  see  here,  nearly  100%  M  cell  degranulate, whereas  knockout  MRGBt  knockout  mass  cell, there's  no  response  at  all  to substance  P.  And  then  besides  the  degranulation, they  also  measured  the  chemokine  release. So  here,  just  show  you  a  couple  examples  of  CCL  two, CCL  three  released  from  cell  degranulation, so  induced  by  substance  P  treatment. So  the  Wilt  M  cell  release  a  lot of  CCL  two  and  CCL  three  upon substance  P  stimulation  whereas  MRGB two  Marcom  substance  B fail  to  induce  CCL  two,  CCL  three  release. And  then  they  also  does  also  inject substance  P  into  the  skins  and see  the  immune  cells  recruitment. So  here  we  use  three  different  group  of  mice, Wil  type  mice,  the  NK  one  narco  mice and  MRGB  two  narco  mice. In  both  WL  type  and  NK  one  narco  mice, and  substance  P  injection  into  the  skin  can induce  a  significant  increase on  immune  cell  recruitment  like  CD  45  positive  cells, neutral  field  recruitment, and  also  monocyll  recruitment  in both  NK  one  Naco  and  wildtype  mice. However,  in  the  MRGB two  Naco  substance  P  fail to  induce  immune  cell  recruitment. So  the  substance  P  promote immune  cell  recruitment  through  our  receptor, but  not  from  this  NK  one  receptor. And  then  besides  injecting just  substance  P  into  the  skin, Dasin  also  used  this  hind  paw  incision  model, which  I  can  show  you  the  actual picture  in  the  next  slide. So  basically,  he  just  made  a  small  cut  into the  hind  paw  and  induce  injury  and  the  inflammation, and  then  using  flowcytomery  to  measure the  the  immune  cell  recruitment from  the  tissues  from  the  hind  paw. And  we  Wild  time  mice,  after  injury, there's  a  significant  increase of  immune  cell  recruitment, whereas  MRGB  two  narco  mice, the  recruitment  are  significantly  reduced. So  just  show  you  the  actual  picture  here. So  this  is  a  incision  model, wild  type  and  knockout  and  hind  paw. And  so  here  the  picture  illustrates this  immune  injury  induced  inflammation is  very  prominent  in  the  wildtype  mice, but  it's  significantly  reduced  in MRGBt  knacko  mice  and  consistently, the  injury  induced  pain  sensation. Here  we  measure  the  mechanical  pain. Also  thermal  pain  has  the  same  phenotype. The  ww  types  show  very  robust or  very  severe  injury  induced  pain. Whereas  a  knockout,  the  pain  is  significantly  reduced. So  suggesting  that  MRGB  to contribute  to  inflammation  and  pain. So  here  just  summarize  this  part  of  the  work. And  so  upon  tissue  injury, the  sensory  nerve  can  release  a  lot  of neuropeptide  like  substance  P  and  activate nearby  cells  through  the  receptor MRGB  two  to  release  chemo  cytokine to  induce  immune  cells recruiment  and  induce  neurogenic  inflammation  and  pain. In  a  parallel  study, the  former  student  Jimmy  actually  also  look  at the  MRG  B  two  in  my  cell  into  the  nonhomergic  age. So  as  you  know,  histamine  can  also  cause itch  during  asthma  allergy. And  so  Jimmy  did  a  side  by side  comparison  between  these  two  major  pathways, mall  degranuation  pathways. And  he  found  upon  the  allergic  reaction  pathways, the  M  cell  actually  release  a  lot  way  more histamine  and  serotonin  on  the  other  hand, if  you  activate  this  muscle  with  MLGB  two  pathways, they  release  less  histamine  serotonin,  but  more  tryptase, and  they  actually  activate  different  set  of  sensory  nerve nearby  so  to  induce  non  hm  alergic  H. So  now  I  want  to  switch  gear  to  talk  about another  biology  we  just  discovered. So  we  found  a  defensin  receptor  in  neutro  field, which  can  prevent  skin  dysbiosis  and  bacterial  infection. And  this  work  is  done  by  a  form  of poster  syntod  and  so  what  is  defensing? So  the  defensing  are  the  antimicrobial  peptidees first  identified  from  frog  skin  in  1987. So  defensing  is  very  important  for  us,  actually. I  can  directly  kill  the  invading  bacteria  and  fungi. And  there  are more  than  50  defensive  peptidees  in  human  mice. And  it's  expressing  the  skins  also  on  GI  tract. And  so  they  can  directly  kill the  bacteria  because  they  can  form this  helix  loop  helix  and  then  insert into  the  negative  charged  bacterial  cell  wall to  make  a  hole  to  kill  the  bacteria. So  people  actually  can  design antibiotics  based  on  the  structure  of  defensings. And  however,  due  to  the  gene  redundancy, because  there's  50  of  them  and  genetic  tools, the  vivo  mechanism  of  defensing  action  is  not  unclear. But  especially  we  don't  know  whether defensin  besides  can  directly  kill  the  bacteria, can  they  actually  also  signaling  our  whole  cells? If  that's  the  case,  what's  a  wholesale  receptor? For  defensins  is  unknown. So  how  to  really  solve  this  problem, there's  50  defensin  genes  in  the  genome, but  they  all  clustered  on  the  mouse  chromosome  eight. So  Symptom  actually  take  effort  to  make a  conditional  knockout  line  but  introduce lake  psite  into  each  end  of  the  defensin  cluster. And  so  this  process  take  about  two  years. So  it's  very  long  process, and  it  turned  out  really  well. So  it  actually  worked. So  by  introducing  the  lax  P  site into  at  the  end  of each  end  of  the  defensive  gene  cluster, and  then  she  crossed  with  a  Caratinosize, specific  cree  called  K  14  cree, and  then  actually  the  deletion works,  that's  pretty  amazing. So  we  can  delete  all  3  million  base  pair  altogether, about  50  defenses  altogether. Showing  the  genomic  PCR show  the  whole  cluster  actually  deleted, so  you  can  have  this  PCR  product  made. And  then  she  also  look  at  the  MRA  for  defensing. So  here,  she  use  control  wild  type  skins or  the  wild  type  skin  infected with  stepors  bacteria  or  Assay. You  can  see  after  infection, there's  a  strong  induction  of  defensing  expression. However,  in  the  defensive  cluster  narco  mice, the  defensing  expression  is  completely  gone. So  here  just  show  the  immuno  staining  of  the  defensins. And  so  she  used  antibody  to  look  at  the  defensins. So  without  infection, there's  not  much  defensing  expression. After  the  Steph  Os  infection, and  you  can  see  it  turned  on. And  however,  in  the  defensins  knockout  skins, there's  not  much  de  peptie  expression  at  all. And  to  make  a  long  way  short  and  Xintong  also  identified another  MRG  member  called  MRGA  two  is defensing  receptor  and  specifically expressing  a  neutral  field. Here  she  just  used  RScope the  red  signal  to  look  at  the  MRGA two  expression  and  use six  G  as  a  marker  for  neutral  fields. And  she  found  about  40%  of  neutral  field  wildtype  Well ti  mice  show  this the  MGA  two  expression  in  neutral  field. However,  in  the  MGA  two  knockout  mice, the  neutral  field  expression  is  completely  gone. Okay.  Then  she  also  did a  neutral  field  degranulation say  essay  for  this  Elastics  release, and  she  found  defenses  can  induce  those  response, uh,  elastics  release  from neutrophil  in  the  wilt  neutropel. However,  in  MGA  two  knockout  neutro  field, the  elastics  release  is  completely  eliminated. So  suggesting  the  MRgA  two  is a  defensive  receptor  expressing  neutral  field. So  a  healthy  skin  can  maintain  a  balance and  diverse  microbiome  on  their  skins, which  can  prevent  bacterial  infection. However,  if  you  have  any  pathological  disturbance, they  will  lead  to  outgrowth  of pathogenic  bacteria  at  the  expense of  other  diverse  camoslls. So  here,  a  uh, into  actually  perform  this  16  S  ribosomal  RA sequencing  for  the  bacteria  on  the  naive  skins, comparing  the  wildtype,  the  defensingnac, and  also  the  defensive  receptor  HA  two  narc  skins. Uh  She  found  on  the  wildtype  skin,  naive  skins, the  diversity  of  bacteria  is  very  high, whereas  in  the  narcot  skins, the  diversity  of  the  microbiome  is  significantly  reduced. And  then  look  at  the  individual  bacteria  strength  uh  uh, she  found  in  the  Wild  time  mice  skins, there's  a  very  diverse  and  balanced  the  bacteria, different  type  of  bacteria. However,  in  the  knockout, there's  expansion  of  pathogenic  bacteria. For  example,  this  green  one, Staph  locus  is  way  expanded compared  to  the  uh  wildty  mice. Suggesting  without  this  receptor  or defensing  and  the  mice  has  this  biosis. So  the  bacteria  composition  is  totally  changed. And  uh  and  beside  the  naive  skin, and  symptom  also  infected  the  skin  by the  exogenous  Sephoris  so  she  can either  paint  the  bacteria  onto the  skin  or  inject  the  bacteria  onto  the  skin, either  wild  type  or  the receptor  narcot  or  the  defensive  narcot. And  in  the  wild  type  skin, you  can  see  the  lesion  sites  cause  bacteria. And  also  the  bacterial  load  in the  wildtype  are  significantly  lower  than  the kakout  animals  many  days  after  the  infection. So  here,  just  show  the  examples,  the  represented  images. So  as  you  can  see  here, the  knockout  after  the  injection, the  bacteria  lesion  site is  a  lot  bigger  than  the  wildtype  mice, whereas  using  the  bio  luminescence imaging  we  found  after  the  infection, both  knockout  showed  a  significant  higher  bacterial  load compared  to  the  wilti  mice. So  this  data  indicate without  defens  or  without  defensing  receptor, the  skin  cannot  fight  well  with  a  bacterial  infection, so  you  have  a  way  severe  infection  afterwards. So  one  of  the  hallmark  of the  sporis  infection  is forming  this  neutral  field  absence. So  here,  Symptom  did the  skin  histology  24  hour  after  the  stporis  infection. So  in  the  Wilt  mice  and  knockout  and she  used  gran  staining to  look  at  the  bacteria  shown  here. And  then  for  the  LG  to  look  at  the  neutral  field  cells. And  as  you  can  see  here, in  the  Wilt  mice  after  stephors  infection, the  bacteria  actually  confined  in a  small  region  and  surrounded  by  neutral  field. So  actually  neutro  field  actually  attack the  bacteria  to  confine  them  into  this  abscess. However,  in  both  defensive  knockout or  the  receptor  knockout, there's  not  much  neutro  aggregation, and  the  bacteria  actually  spread  in  a  large  area. So  that's  why  you  have  a  larger  the  larger  lesion  size shown  here,  higher  bacteria  load. And  here  just  show the  quantification  of  neutral  field  recruitment. And  you  can  see  here,  Wilt  mice  has  a  lot  of way  more  neutral  fuel  recruitment where  after  the  infection, whereas  in  the  knockout, the  neutro  field  recruitment  is  significantly  reduced. So  both  defensing  and  receptor  really mediate  neutral  field  recruitment  and  abscess  formation. So  mechanistically, we  found  after  the  bacterial  skin  infection, the  neutro  f  actually  can  release  cytokines,  chemokines. Here,  we  just  show  you three  different  examples  like  one  Beta, CXCL  one  or  two, and  we  found  both  in the  nac  in  the  wildtype  after  infection, all  these,  chemokine  cytokine  are significantly  increased  release  by  the  neutral  field. And  then  the  narco those  narco  the  cytokine  chemokine  release are  significantly  reduced, and  so  they  mediate the  cytokine  and  the  chemokine  release. So  here,  just  summarize  this  part  of  the  work. So  we  think  in  healthy  skin, there's  a  low  level  of defenses  level  of  defensing  and  they  actually keep  this  diverse  and  balanced  microbiome maintained  on  the  skin  on  the  healthy  skins. And  there's  also  some  skin  resin  neutrophil, keep  the  healthy  skin  intact. However,  after  the  bacteria  infection, and  this  balance  is  disturbed, and  then  there's  upregulation  of  defensing  increased. And  then  they  actually  activate skin  residn  neutropel  through the  MRGA  two  receptor,  and  they  release  a Uh,  interlooking  one  Beta  or  CX  one  or  two, and  they  can  recruit  more  neutrophil  infiltrate  into the  area  to  form this  neutrophil  abscess  to confine  the  bacterial  infection. So  without  the  defensing  and  without  the  receptor, and  this  process  is  disturbed, and  then  you  have  way  worse  infections. So  here,  I  just  want to  show  you  what  we're  going  to  do  next. So  as  I  mentioned, we  found  a  muscle  receptor,  specific  receptor, and  its  function  receptor  for many  neuropeptides  like  substance  P and  play  important  role  in the  neurogen  inflammation  and  paan  H.  And  now  we  are working  on  the  role  of  the  receptor or  lls  in  the  ischemic  stroke, uh  and  also  in the  fibromyalgia  pain  and  multiple  sclerosis. So  right  now  we  are  using  again, using  mouse  model  for this  disease  to  look  at  the  muscle  and  muscle  receptor. And  hopefully  in  the  future, we  can  develop  a  small  molecule  antagonist  blockers to  treat  many  of  the  disease. In  fact,  the  phase two  clinical  trials  is  going  on  right  now  to testing  some  of  these  conditions using  the  MRGX  two  antagonist. And  for  the  MRG  A  two,  uh,  project. So  again,  this  is  a  defensin  receptor, and  now  we  are  moving  into  the  brain  to  look  at the  brain  infection  like  meningitis and  see  whether  defensin  play  a  role  there. And  also,  we  want  to  know  whether the  defensins  and  MRgA  to  play  a  role  in  psoriasis. Is  a  condition  where  you  can  see a  strong  ab  regulation  of  defenses  in  the  psoriasis. So  as  mentioned,  this  receptor  play  a  diverse  role  in many  basic  biological  process  and  also  disease  from  pain, ache  and  innate  immunity. And  so  we  only  dorphanize  half  of  this  gene  family, and  still  many  of  them, we  don't  have  clue. We  don't  know  where  the  expression  and  the  ligand, so  there's  still  a  long  way  to  go. And  thank  you  very  much  for  your  attention. So  here  is  the  people who  have  done  all  the  works.  Thank  you. Pricing.  Yeah.  And  plastic  word  thank  you. Two  related  questions. Yeah.  Sorry  if  I  missed  this. But  given  that  X  four  can respond  to  bilirubin  as  well  as  bile  acids. What  is  the  evolutiary  advantage  to  inducing a  niche  response  to  when  there's,  like,  liver  distress? Yeah,  so  we  don't  know whether  this  receptor  have any  physiological  roles,  right? So  recently,  we  published  a  paper showing  this  also  receptor  for  fossil  group. So  we  identified  many  fossil  group  drugs can  activate  this  receptor. It  turned  out  adding  fossil  group  is  a  common  way to  in  drug  development  to  induce  water  solubility. So  people  are  actually  adding intentionally  adding  phos  group into  difficult  drugs  to  dissolve  in  water, but  at  the  same  time,  they  each  a  a  side  effect. So  we  found  those  each  side  effect actually  caused  by  this  receptor  also. And  uh  but  from  that, we  actually  find  some  other  lipid indgent  lipid  phospholipid  like geno  diphosphate  can  activate this  receptor  at  a  very  low  EC  50,  like  nanomolar  range. So  maybe  that  can  give  us  some  hint  whether this  receptor  can  sensing more  indulgent  phospholipid  or  something. So  we  are  working  on  that.  Yeah. Yeah.  Yeah,  go  ahead. Do  you  have  second  question  or  you  sort  of  answered  it, the  neurons  that  Express  por  are they  known  to  respond  to  any  other  stimulus. So  it  seems  like  they're  Yeah. Thank  you. Go  ahead.  Maybe  same  point. From  your  data  is  though the  Billy  Rubin  neurons  Billy  Rubin  are  multimodal. Yeah.  So  I'm  wondering  if  you  looked  at other  ligands  in  terms  of  what  else  might  act? Yeah,  so  these  neurons  can  activate  bilirubin  bio  acid, some  other  itchy  substance. And  in  mice,  it  turned  out there's  three  different  itchy  sensing  neurons, and  they  called  NP  sorry  P  one,  NP  two,  MP  three. And  so  in  mice,  there's  a  small  subset  of  each  neurons. However,  in  human  is  a  little  bit  complicated. Maybe  they  are  more  converge  into the  one  large  subset  and one  neuron  can  respond  to  multiple  itchy  substance. That's  possible,  too.  And  somehow  the  mice  has, like,  more  dedicated  each  neuron  now. It  seems  that  those  neurons  primary  sensory  neurons be  mechanical  sensor.  Too. Yeah,  yeah,  they  do  express  PSO, but  people  actually  in  mice,  at  least, identify  mechanical  sensitive  neuron,  too, mechanical  sensitive  mechanical  H  neuron, actually,  yeah.  Yeah,  yeah. Yeah,  go  ahead.  So,  I  mean, first  off,  thank  you  for  coming  to  speak  with  us. Yeah.  I  learned  a  lot. I'm  kind  of  curious.  I  know  at  the  end  you  touched  on antagonists  for  MRD  for  fibromyalgia.  Yeah. Two,  sorry,  VT. Yeah.  So  I'm  kind  of  curious. Do  you  think  there's,  like,  a  hyperepression going  on  in  those  patients  or  what? Yeah,  so  the  so this  receptor  recall  is  called  MRgX  B  two, sorry,  for  the  mass  cells. And  as  I  show  you  at  the  RTPCR, the  receptor  is  already  expressed very  high  level  in  those  cells. And  we  don't  think the  expression  in  particular  cell  going  to  go  up. And  more  because  they're  already very  high  expressing  genes. So  maybe  the  number  of  new  mall  can increase  or  they  aggregate,  that's  possible. Yeah.  People  actually  has the  data  showing  the  fibromyalgia. We  have  our  collaborator  have  poplic  data  showing the  fibromyalgia  patients  skins actually  has  a  lot  of  mass  aggregates. So  yeah,  so  there's  many  companies  working on  this  receptor  and  tagons  right  now.  Yeah. Thanks  for  the  great  talk.  I  was curious  about  the  defensive. Yeah.  Here  what  cell  type  they're  coming  from? I  think  they're  signaling to  the  neutrophils,  is  that  right? Yeah.  So  basically,  we  show  there's  two  actions. Other  people  show  there's a  direct  killing  action  to  the  bacteria, but  now  we  show  they  can  activate  the  neutral  field. And  they  mainly  from  the  keratino  site. So  that's  Beta  defens. There's  also  Alpha  defens. Alpha  defenses  is  more  in  GI  track. They  are  made  a  different  type  of  cells. And  so  we  found  actually the  Mina  cells  also  make  defensins. So  that's  why  we  are  looking  at  ndagitis  right  now. Yeah.  Any  other  questions? Yeah.  Thank  you.