Finding Balance in Iron Metabolism

April 28, 2022

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April 28, 2022

Yale Pathology Grand Rounds

Karin Finberg, MD, PhD

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00:00Today's speaker is Karen Feinberg.
00:03Doctor Finberg needs no
00:06introduction introduction.
00:08However, we have several new
00:10members in the department,
00:12so for their sake I will
00:15introduce Doctor Finberg.
00:17She's been at Yale for a long, long time.
00:21She came to Yale to do her
00:26undergraduate education and she
00:29graduated BS Magna *** laude.
00:32And then Doctor Feinberg stayed on to
00:36do an MPhil, followed by an MD PhD.
00:42She went on to Mass General Hospital to
00:46do a residency in clinical pathology,
00:50followed by a fellowship in
00:53molecular genetic pathology at
00:55Brigham and Women's Hospital.
00:57So after that, however,
00:59it looks like Doctor Feinberg's interest's
01:02basic interest was in iron metabolism
01:06and she did a research fellowship
01:09at BWH and then she was recruited.
01:13At Duke University,
01:15where she spent another four years building
01:19her research until thanks to Doctor Morrow,
01:23Karen was recruited here at Yale in
01:282013 to continue her research work.
01:33Doctor Feinberg's track record
01:36track record of Excellence was
01:40established early on during her
01:43undergraduate education when she was.
01:47Became member of the Phi Beta Kappa Society
01:52for Excellence in Humanities and in Arts.
01:56But at Yale College,
01:59she also became a member of
02:02prestigious Honor Society,
02:04Alpha Omega Alpha Society
02:07for medical students.
02:09She received a Yale MD PhD Alumni Award and
02:13she received two Young Investigator Awards,
02:17one in 2006 from the Association
02:21of Molecular Pathology.
02:23While she was still a fellow and.
02:26Another in 2013 from International
02:30BioWare and Society.
02:33Doctor Finberg has been a Co
02:37investigator on several grounds at Yale,
02:40and I will not enumerate them.
02:43She's currently associate director
02:45of tumor Profiling Lab and she
02:48plays a seminar where she played
02:51a Seminole role in implementing
02:55clinical next generation sequencing.
02:58She is associate director
03:00of Yale Molecular Genetic.
03:03Pathology fellowship and assistant
03:05Director of Education for the Yale,
03:08MD, PhD program.
03:10Her research focus is primarily primarily on
03:14molecular basis of inherited iron disorders.
03:19Mechanisms of systemic iron regulation,
03:22and physiological consequences
03:25of iron deficiency anemia.
03:28So, with that I'll let Doctor
03:31Feinberg start for those of you.
03:33Who would have questions at
03:35the end of the talk?
03:37Feel free to unmute yourself
03:40and ask questions.
03:41Those who are on zoom.
03:43Thank you.
03:45So thank you Doctor Prasad and I wanted
03:47to say that it's a particular honor
03:50to follow Doctor Keshishian who spoke
03:52in ground grand Grounds last week.
03:54So Doctor Cashiering was one of
03:56my mentors when I was an MDP.
03:57She sitting here and he's actually the
03:59first person that really told me to think
04:01seriously about a career in pathology,
04:03so perhaps he's listening today.
04:04I hope so. I have nothing to disclose.
04:08So today we have a diverse audience
04:11and our department, so I'm going to
04:13try and cover a lot of ground. My talk.
04:16I'll start with an introduction to
04:18the iron regulatory hormone hepcidin,
04:20and talk about its dysregulation and
04:23human genetic disorder of iron loading.
04:25But I'll move on to the condition iron
04:28refractory iron deficiency anemia, or Rita,
04:30a genetic disorder of hepcidin, excess.
04:34Then we'll move to new insights into
04:36mechanisms of iron mobilization
04:38from the liver.
04:39From my laboratory work here and
04:41finally some collaborative studies.
04:43I've done it.
04:44Yeah,
04:45looking at the physiological consequences
04:47of iron deficiency beyond anemia.
04:50So let's start with iron regulation.
04:53As this audience is well aware,
04:56the maintenance of systemic iron
04:58balance is really critical for health.
05:002 little iron impairs of production
05:02of many essential proteins,
05:04most notable of course.
05:05Hemoglobin leading to anemia,
05:07and I'll remind you that iron
05:09deficiency anemia is the most common
05:12micronutrient deficiency worldwide.
05:14About 80% of the global
05:16population has low iron stores,
05:19as estimated by The Who,
05:21and about 30% of the global population
05:23has overt anemia due to iron deficiency.
05:27Conversely,
05:27too much higher the problem for the body,
05:29because iron causes oxidative damage
05:32to tissues and this iron overload
05:34can be acquired through chronic
05:36blood transfusions and also in
05:37a variety of genetic disorders,
05:39including for example HFE
05:41hereditary can chromatolysis,
05:43as well as several iron loading remias.
05:48Iron Physiology involves a complex
05:50interplay between multiple organs.
05:52Iron is absorbed in the duodenum and
05:55it's transported into the Plaza, where it
05:57finds the carrier protein transparent.
05:59Iron Dentist delivered to the bone
06:01marrow for red blood cell synthesis.
06:03These red cells circulate and when they age,
06:05they're phagocytosis by
06:06macrophages in the spleen.
06:08This allows the iron to be reclaimed
06:11from hemoglobin and exported
06:13back into circulation to support
06:15further rounds of auricular crisis.
06:17Iron, of course,
06:18can also be used by other organs,
06:20such as the heart, muscle,
06:21pancreas and of course iron is needed
06:23for the fetus during pregnancy.
06:26The liver is the body's main iron Depot,
06:29the main site of iron storage.
06:33And daily we actually lose very
06:35little iron from the body.
06:37We have small losses from shedding of
06:38the lining of the of the GI tract.
06:40The Gu tracks the skin.
06:42And of course menstruation and women.
06:44But there's no known regulated mechanism
06:47for iron excretion from the body.
06:49As a result,
06:51it's become clear that systemic
06:53iron balance is regulated at the
06:55level of intestinal absorption.
06:57And so I thought,
06:58inspired by Doctor Kashgari
06:59and I show a little history.
07:01So this is George H.
07:02Whipple,
07:03who was a pathologist,
07:04actually a Yale undergraduate as well.
07:07He's the whipple of Whipple's disease,
07:08not procedure,
07:09and he shared the 1934 Nobel Prize
07:13for discovering it's concerning
07:14liver therapy in cases of anemia.
07:17This is a publication from his
07:19group from 1943 in the Journal
07:21of Experimental Medicine,
07:21where they looked at radioactive iron
07:24absorption by the gastrointestinal
07:27tract of dogs.
07:28And he made some summer lovations that
07:31the gastrointestinal mucosa accepts
07:33iron readily when the Iron Reserve
07:36stores are depleted by chronic anemia,
07:38but in a plethoric state when there is,
07:40there is very little absorption
07:42of iron and the body has no ready
07:45means of disposing of surplus iron.
07:47The body can protect itself against a
07:51large accumulation of iron with the body,
07:53which can cause damage to important organs,
07:55as in hemochromatosis and
07:57Mediterranean anemia.
07:57The traditional name for a historical
08:00name for beta thalassemia.
08:02And the mechanism of this acceptance
08:04or refusal of iron is of great interest
08:07to physiologists and physicians.
08:08So it took a long time for that
08:11mechanism to be discovered,
08:13but in the early 2000s it became
08:15clear that the hormone called
08:17hepcidin is the central regulator
08:19of systemic iron balance.
08:20So upside Nessa,
08:22small peptide hormone produced
08:23by hepatocytes you can detect it
08:26in both blood and urine,
08:28and hepcidin regulates iron balance
08:30primarily by acting it to sites.
08:32So upside and acts on enterocytes to
08:35limit the absorption of dietary iron.
08:37And hepcidin also acts on macrophages
08:39to limit iron export from these cells.
08:41This is the iron that has been
08:44reclaimed from senescent red blood cells.
08:47To have side and limits the
08:49recycling of iron stores.
08:50And molecular level have sided mediate
08:53these effects by binding to fair portion,
08:55which is a cellular iron exporter
08:57present on the basolateral
08:59surface of enterocytes and also on
09:01the plasma membrane of macrophages.
09:03Have side in binding to Fairport
09:05and causes ferroportin,
09:07endocytosis and degradation in lysosomes.
09:10Additionally more recent work on
09:12from the Group of elements has
09:14shown that upside and actually
09:15also includes the Fairport and
09:17transporter blocking iron transport.
09:21Have side an expression is modulated
09:23in response to liver iron stores.
09:25This is the basic regulation in the normal
09:27state so that when body iron stores decrease,
09:30have side and production is reduced and
09:32this allows iron and iron absorption from
09:35the intestine promoted as well as the iron
09:38release from macrophage stores to proceed.
09:41The net result here of course is to maintain
09:43the level of iron in the circulation to
09:46maintain the supply of iron for replaces,
09:48the net effect being the prevention
09:50of systemic iron deficiency.
09:52And conversely,
09:53when body iron stores increase liver
09:55increases hepcidin production inhibiting
09:57further dietary iron absorption and
10:00inhibiting iron released from macrophages.
10:06So genetic defects impacting the website
10:09in Fairport and access underlie the major
10:12inherited primary iron overload disorders.
10:15There are five major disorders shown here.
10:18I'm going to focus first on these.
10:20These first three categories, SHFE related,
10:23hemochromatosis juvenile hemochromatosis,
10:25and TFR 2 related hemochromatosis.
10:29So all of these disorders
10:31are recessive disorders.
10:33And characterized by similar patterns
10:34of iron loading in the liver,
10:36heart, and endocrine glands.
10:38And the stylar iron accumulation
10:41pattern is prankowl.
10:43The gene products that are mutated
10:45in these forms of hemochromatosis H,
10:47Fe HJV or Hematoxylin.
10:49HAMP, which is the hepcidin gene
10:53itself or transparent receptor
10:542A homologue of the transferrin
10:56receptor on erythroid cells.
10:58All of these gene products are are
11:01acts in in parasites to promote
11:03upside and transcription,
11:05and I'll show you in a second the
11:07other class of iron overload disorder
11:10in this table are you Lisa disorders?
11:13Between mutations in Fairport and itself
11:15and there are two different forms.
11:16They show autosomal dominant
11:18inheritance and the features differ
11:20depending upon the mutations or gain.
11:21A function or loss of function mutations.
11:24But I want to mostly focus on these non
11:28Fairport and types of iron overload.
11:31Because they all seem to converge on a
11:33common signaling pathway in the liver,
11:35so bone,
11:35refrigerated protein or BMP SMAD
11:37signaling is the major signaling pathway
11:39promoting upside and transcription.
11:41Perhaps sites in this pathway.
11:44The BMP 6 ligand binds to receptor complex.
11:47Promote the phosphorylation of
11:49intracellular SMAD proteins which
11:51translocates to the nucleus,
11:52promoting the transcription of
11:54upside and other BMP target genes.
11:57The light interior BMP 6 interestingly
11:59appears to be produced by liver
12:01sinusoidal endothelial cells
12:02in response to iron levels.
12:04Although the mechanism by which
12:06liver sinusoidal endothelial cells
12:08sense iron to express the MP,
12:10six is still under investigation.
12:13Hema Jubelin but one of the gene
12:16products mutated in juvenile
12:17hemochromatosis is the GPI anchored
12:19protein is the coreceptor for BMP's
12:22and hepatocytes and hemogoblin
12:23augment signaling to the BMP pathway.
12:28HFE, the well known hereditary can
12:31comatose this protein and transparent
12:33receptor 2 are transmembrane proteins
12:36that interact with the classic
12:38transferrin receptor TNFR 1 to form
12:40what is believed to be an iron sensing
12:42complex that responds to levels of
12:45transparent bound iron and circulation
12:47and through and through the sensing,
12:49modulate herbicide and transcription.
12:52It's thought that perhaps these
12:53proteins all participate in a
12:56giant so-called super complex,
12:57that that still remains to
12:59be fully demonstrated,
13:00but it seems likely that HFB and TFR 2
13:03signaling also impact on the in peace man,
13:06the B and PEACEMAN pathway.
13:09So I'd like to move on now to from a
13:12disorder disorders of hepcidin deficiency.
13:15In the previous slide to disorders
13:18of upside in excess.
13:19So when I started my postdoc
13:22with Nancy Andrews,
13:23she marked Fleming and Matt
13:25Henry at Children's Hospital.
13:27Boston had collected DNA from a
13:30number of children and never kindreds,
13:33in which the children had iron
13:35deficiency anemia that was
13:37refractory to oral iron therapy.
13:39And they called this condition a
13:41red for this. For this phenotype.
13:43The key clinical features were congenital,
13:46hypochromic,
13:46microcytic anemia and extremely low.
13:50Erythrocyte mean corpuscular volume,
13:51sometimes down into the 40s.
13:53So extremely low.
13:54Very low, transparent saturation.
13:56A failure to respond to oral iron.
14:00And a sluggish,
14:01incomplete response to intravenous
14:03or intramuscular iron and looking
14:05at the pedigrees,
14:06particularly this first pedigree from Turkey.
14:08With consanguinity,
14:09you can appreciate that transmission was
14:12compatible with recessive inheritance.
14:14Here's the typical blood smear from
14:16one of the affected individuals,
14:18showing classic signs of asphere
14:21iron deficiency anemia.
14:23So when I came to Nancy's lab and Eliza
14:26assay to measure hepcidin levels in patients,
14:30blood and urine had just become available
14:33and looking at the phenotype individuals,
14:35we were suspicious.
14:36The phenotype of these individuals
14:37and we were suspicious that they
14:39had a defect in hepcidin regulation
14:40and sure enough we found that have
14:42side levels in these individuals
14:44were inappropriately elevated.
14:46So this of course explains the
14:48pathophysiology of this disorder.
14:50Normally an iron deficiency have
14:51side and level should be low,
14:53but in these in these individuals
14:54who had decreased iron stores,
14:56there have a side,
14:57and it's paradoxically increased.
14:59So it's explains both their failure to
15:01respond to oral iron as well as their
15:03failure to respond to intravenous
15:05or intramuscular forms of iron.
15:06Because these forms of iron require
15:09processing by macrophages before
15:10this iron can be exported into the
15:12circulation for use in erythropoiesis.
15:16So this is this was back in 2007
15:21and before exome sequencing was,
15:24it's cheap as it is today, and so I did.
15:27Traditional linkage analysis,
15:28mapping to map the phenotype
15:30to region of chromosome 22.
15:32This was a region of about 100
15:34megabases many genes to sift through.
15:37And as I was sifting through a particular
15:39gene that came to our attention,
15:41which is a gene called tempra 6,
15:42also known as matriptase 2.
15:45So this encodes a type 2
15:48transmembrane serine protease.
15:49Transmembrane serine protease
15:516 is now it's official name.
15:53That was known at that time to be
15:56primarily expressed in the liver,
15:57and had been found to be localized
15:59to the plasma membrane when
16:00overexpressed themselves at that time,
16:02there were only two publications
16:04on this protein.
16:08Structurally, this protein consists
16:09of a short intracellular region,
16:11a transmembrane domain,
16:13and a large extracellular
16:14domain with several motifs,
16:16including most notably AC,
16:17terminal, serial, protease, domain.
16:21What caught my eye was in the abstract
16:24book for the 2007 ASH Annual meeting,
16:26where the group of Ernie and
16:28Bruce Boiler reported this.
16:29Mouse munit temper 60 and new new mutant,
16:32which is a mouse that you can see from
16:35this picture, has hair on its head,
16:36but it's all for the rest of his
16:39body called the Mask Mutant.
16:41The baldness relates to iron deficiency.
16:43It's cured by feeding iron and they show
16:46that these mice of course they they.
16:47They map the temper 16 and the strain and
16:49they showed that this was a recessive.
16:51The phenotype where the mice had
16:53iron deficiency anemia due to
16:55impaired and absorption and looking
16:56at the liver of these mice they had
16:58elevated have side messenger RNA,
17:00so obviously this was an excellent
17:02candidate for the erythema type and I
17:04went on to look at 10% as a candidate
17:06in kinders with a Rita and we found a
17:09number of mutations across the molecule,
17:11classic,
17:12frameshift mutations,
17:13nonsense mutations and a
17:14variety of missense mutations at
17:17evolutionarily conserved residues.
17:20So. Distinguishing Arita from other
17:24required forms of iron deficiency anemia
17:27cannot always be so straightforward.
17:30I worked with Matt Heaney at
17:32Children's Hospital Boston,
17:33develop some diagnostic
17:34algorithms for the disorder,
17:36and I want to point out one of the
17:37key aspects of these algorithms
17:39is to consider how patients
17:40respond to an oral iron challenge.
17:42So you really want to look at how
17:44their serum iron levels change after
17:45you give them iron in the short term.
17:48Short term,
17:49after a fire administration
17:50to help differentiate.
17:51This is truly an absorptive.
17:52Effect before proceeding.
17:54Then at that time you know when genetic
17:58testing was a little more expensive.
18:00You know we we weren't going to think about
18:02testing temper 6 immediately up front.
18:04I think now with Exos,
18:05a little
18:05bit of a different story.
18:08I also collaborated with Matt and Mark
18:11Fleming to look at hepcidin levels
18:14in patients with Arita and how they
18:17compared to other patients with iron
18:20refractory anemia that wasn't couldn't
18:22be attributed to 10 per six mutation.
18:24And indeed you know, as expected,
18:26the hepcidin levels in patients with
18:28temper 6 mutation based on our original
18:30court where again seemed to be increased
18:32but there was some overlap with patients
18:34who did not have a temper 6 mutation.
18:38And so we looked more closely
18:40at various laboratory indices,
18:41and you can see with these
18:43receiver operating curves the red
18:44curve here is to have side in.
18:45This is a fairly good job of predicting
18:48temper 6 mutation status in patients
18:50with chronic iron refractory anemia,
18:53but better indices turn out to be
18:55indices where you normalize the
18:57helpside into either the serum iron
18:59or the the transparent saturation.
19:03So of course I wanted to actually
19:05figure out how society was working
19:07to regulate upside in production.
19:09The Group of Cleric and Michaela did
19:12some beautiful invitro work showing
19:14the temper 6 cleaves Hema, Julian,
19:16the BMP coreceptor from the plasma membrane.
19:19This is the gene product I showed
19:20you a few minutes ago with one of
19:22the genes that's mutated in juvenile
19:25hemochromatosis and I went on and
19:26now smodels to show that mice lacking
19:28temper 6 show excess signaling through
19:30the BMP pathway that is dependent
19:32upon the presence of hemogoblin.
19:39And then wait additional studies looking
19:41at genetic loss of temper sticks and
19:44other mouse models of clinical iron
19:46overloads where iron overload is due to
19:49have side insufficiency in particular,
19:51predatory tosis due to HF mutation and also
19:54non transfusion dependent beta thalassemia.
19:57I won't get into the mechanism there
19:59but let's just say briefly that inherent
20:01in a congenital iron loading anemias
20:03have signed news also low promoted
20:05which promotes iron loading and our
20:07group as well as the canvas shell.
20:09Group demonstrated that indeed,
20:10temper six loss is a way to raise
20:14upside and limit iron loading in
20:16in these iron loading disorders.
20:19So approaches to modulate temperance
20:21expression or under investigation.
20:23So in addition to just doing the
20:25classic knockout experiments,
20:26groups have looked at small interfering RNA
20:29S RNA formulated in lipid nanoparticles,
20:31as well as antisense oligonucleotides.
20:33The target temper 6 M RNA.
20:36Here's here's a diagram showing the
20:38antisense approach and the commentary
20:40Rd on that paper and the idea here
20:43of course is to cause degradation of
20:45the temper 6 M RNA with these oligos.
20:48Lose temper 6 from the plasma membrane
20:50of these cells and now promote signaling
20:52through these pathways to elevate have side.
20:55Now I'll point out that currently
20:57temper 6 antisense oligos,
21:00specifically those that have been
21:02caught conjugated to a liver specific
21:04ligand and a settled Galactus demeanor.
21:06Galac are in phase two clinical trials
21:09for humans with pathological media.
21:12I also want to point out that you know,
21:14I showed you the complex CMP pathway earlier,
21:17and while multiple proteins in that
21:19pathways might be considered path
21:21possibilities to modulate upside
21:23and expression,
21:24I want to point out that temper 6 is is
21:26a particularly attractive target in my
21:27mind because unlike many of the other
21:29proteins I showed you in the BMP pathway,
21:31this is a liver specific gene.
21:34So hopefully you could reduce
21:35a lot of off target effects.
21:39So I'd like to move on now and
21:41talk about new insights we've
21:43made into the mechanisms of
21:45iron mobilization and deliver.
21:50So from what I've told you so far,
21:51have sided therapies would seem like.
21:55Reasonable approach to limit iron loading,
21:58but one of the problems I see with those
22:00types of therapies is they don't really
22:02address the problem of iron loading.
22:04It's already present once,
22:05once in a dual mileage is loaded,
22:07lowering raising their upside level is not
22:09going to help eliminate the iron the iron.
22:11This morning the body,
22:13you know hereditary hemochromatosis sewers
22:15are classically treated by phlebotomy.
22:17Most patients can tolerate slotomania,
22:19although it's definitely inconvenient
22:21to have to be closed on a regular
22:24basis throughout your life.
22:26And although this is the classic therapy,
22:28the mechanisms that promote
22:29our immobilization are really
22:31not well understood at all.
22:33So this was an area I was
22:35interested in exploring.
22:36What are the mechanisms that regulate
22:37the release of iron from the liver?
22:41And I became more intrigued by this area.
22:46When? With these this work from the
22:49Group of Joe Mancius at Harvard and also
22:53group from Novartis characterizing a
22:55role of protein nuclear color receptor
22:58coactivator 4 previously thought
22:59to be a transcriptional regulator,
23:01a new role in the regulation of ferritin,
23:04trafficking within cells and
23:06specifically in cancer cells.
23:08These groups showed that NC A4 is a cargo
23:12receptor that that shuttles the iron
23:15transport complex ferritin to the lysosome.
23:17We're fair to him, can be degraded,
23:19and the iron can be released
23:21into the cytoplasm,
23:22potentially made available for
23:24export cancel as well.
23:26We were interested in this gene
23:27actually was interested in gene
23:28before these papers came up,
23:30because I found it in some micro experiments.
23:32Looking at, you know,
23:33M RNA from livers of mice when iron
23:36overload versus iron deficiency.
23:38So Jay Lee,
23:40who was a phenomenal Xpath
23:41graduate student in my lab.
23:43He graduated last year as well as Larissa.
23:45Loads of Oscar Research Assistant
23:47lab decided to really try and
23:49define if insula forward had a role
23:51in iron mobilization,
23:53the mobilization of iron from ferritin
23:55stores specifically in the liver.
23:58So to do this,
23:59we utilize chemically modified srna.
24:02Since the galnet conjugated srna,
24:04the targets have had ascites to lower NC
24:074 expression to participates in vivo,
24:10and this,
24:10of course,
24:11was after thoroughly validating
24:13these sarnas and invitro.
24:14Before we started the annual work.
24:18So we looked at the responses of
24:20animals that were treated with
24:22either vehicle luciferase control
24:24or end to a forward targeting
24:26Sir and A and then subjected to a
24:29large volume for botany and then
24:31looking a week later to to see
24:33their physiological responses.
24:34So would the srna we see we
24:37obtained excellent knocked out
24:39of entry for in the liver.
24:41And you can see that one week after
24:44spodni mice treated with vehicle
24:47control or luciferase control.
24:49So to mark reduction hepatic Iron store,
24:51the iron was exciting,
24:53but in mice with Intuit for knockdown,
24:55we didn't see that massive reduction
24:57librarian stores and similarly
24:59looking at ferritin protein levels,
25:02we see that compared to the baseline
25:04state mice treated with vehicle
25:06luciferase controls showed marked
25:08reduction to ferritin protein,
25:09which is not seen in mice with
25:11ensued 4 knocked down.
25:13And we did further characterization
25:14to just make sure that ensued.
25:16Mice within two four knockdown
25:18showed similar degrees of anemia,
25:19transferrin,
25:20saturation,
25:20and have silence suppression
25:22after full bodies.
25:23So these other factors we couldn't
25:25explain the difference in the liver
25:27iron loading phenotype that we observed.
25:29So and so if we're appears to be
25:32limiting hepatic remobilization.
25:34And this led us to question,
25:35you know how is endogenous and two
25:37for activity regulated and what's
25:39the stimulus for iron mobilization?
25:43So we then we just shifted to
25:45work and hepatoma cells have the
25:47cities are have three hepatoma
25:49cells and we treated them first
25:51with a chelator just proximity DFO.
25:54And we found that this caused an
25:57upregulation inside for a messenger RNA.
26:00We realized in these experiments,
26:02not only is DFO keliher,
26:04but it's also a stabilizer of
26:07hip hypoxia inducible factor.
26:09And we treated hepatoma cells with
26:11other chemicals known to stabilize
26:13hip calcium chloride and demog
26:16and observe similar upregulation
26:18in institute for expression.
26:20So you remember that the
26:22poor little hydroxylases,
26:23the HIF prolyl hydroxylases?
26:26Or oxygen and iron dependent enzymes
26:29that promote the degradation
26:31of health HIF alpha subunits.
26:33So in the normoxic and iron sufficient state.
26:38HIF alpha subunits undergo post
26:40translational modification and targeting
26:42to the proteasome for degradation.
26:44But, in the hypoxic or the iron,
26:47sufficient insufficient state.
26:49For example, in the setting of chelation.
26:52Hips subunits alpha subunits are
26:54available to translocate to the
26:56nucleus where they can heterodyne
26:58rise with the constitutive beta
27:00subunit to criminal gene expression.
27:02I'll remind you that they're both.
27:04They're both the hip 1A and a hip 2A subunit.
27:07So we wanted to investigate if
27:09the answer for induction wouldn't,
27:11if one HIF dependent process.
27:13So using end to if we're knocked down.
27:15Sorry using hip knockdown experiments
27:19we were able to show first that in the
27:21absence of knockdown we see a marked
27:24induction of interferon chelation.
27:26The treatment with the FL chelator
27:28and this induction is abrogated
27:31by knockdown of 51 or 52,
27:34particularly the double combination together.
27:37Jade went on to do some data mining.
27:39We found a data set from the lava Peter
27:42Ratcliffe in the UK who had done a
27:45GMY chip seek experiment in hepatoma cells.
27:48These are hep 3G hepatoma cells
27:50under conditions.
27:51Hypoxia and Jade was able to
27:54identify in their data.
27:56It hits one binding site actually hit
27:58one hit two and HIF 1 beta binding site,
28:01about 1.5 KB upstream of the
28:04first exon and two A4.
28:05So this LED us to a model in which
28:08the hip one transcription factors
28:10under HIP stabilizing conditions.
28:12So either iron deficiency or hypoxia promote,
28:15and so if we're transcription
28:17increasing the supply of this protein
28:19to participate in the ferritin,
28:21not for the process described
28:23by the Mancias group,
28:25allowing ferret to be degraded.
28:27An iron to be made available for use
28:29and perhaps exported from the South.
28:32So I think this has been relieved,
28:33revealed a pathway with some
28:36potential for our immobilization.
28:37I think whether this could be capitalized
28:39in iron disorders remains to be seen,
28:41but could be very interesting
28:42to investigate further.
28:46So finally I'd like to move on to some
28:50collaborative work I've done here,
28:52switching now to thinking about
28:54the physiological consequences,
28:55iron deficiency beyond anemia,
28:57and I'll talk about two collaborations.
28:59One is looking at the relationship of
29:01iron deficiency to platelet counts,
29:03and the 2nd is the relationship of iron
29:05deficiency to production of the hormone
29:08fibroblast growth factor 23 or FGF 23.
29:14So as as many in the
29:16audience are probably aware,
29:18iron deficiency media is often associated
29:20with elevated platelet counts.
29:22In humans. This is a agel observation.
29:27I went back and looked at the
29:28platelet counts and the patients with
29:29a ride up and we've been focusing
29:31on their red cell phenotypes.
29:32Not thinking so much about their playlists,
29:34but indeed if you look graph their
29:36hemoglobin levels versus their platelets,
29:38you see a really nice inverse
29:41correlation between these parameters.
29:45And so we wondered if we could
29:47capitalize on the temper 6 knockout
29:49model a mouse model of chronic iron
29:51deficiency anemia to gain insight
29:53into the underlying mechanism.
29:54So you might just ask,
29:55but why not just feed mice and you know
29:57regular mice and iron deficient diet
29:59and induce iron deficiency anemia.
30:00It's actually quite technically
30:02challenging to do that.
30:03Mice have much such small daily iron needs,
30:06and there's so much iron available
30:07in their caging in their bedding.
30:09It's very hard to conduct studies of
30:13in which iron deficiency anemia is.
30:15Consistently induced through
30:16dietary means alone.
30:18So This is why we're interested in
30:20using the mouse model where we had a
30:22genetic form of iron deficiency anemia.
30:24We confirmed that the temper 6 knockout mice.
30:27Of course,
30:28as we move previously had low hemoglobin,
30:31low MCV but also elevated platelets
30:33and we also confirmed their
30:35public plate elevation by facts.
30:37This was actually quite important.
30:38Those of you who remember your lab
30:41medicine know that microsites so small
30:43erythrocytes can easily miscounted
30:45for platelets by cell counter.
30:47This is a particular issue for
30:49mice where the red cell MCV is
30:51much smaller than the humans.
30:53So we confirmed the plate elevation.
30:55And here you can see the
30:56peripheral blood smears,
30:56and I think you can appreciate even
30:58on the temper 6 knockouts where
31:00it increased number of platelets.
31:04So to make sure we could observe plate
31:08elevation in mice with normal hepcidin
31:10regulation we collaborated with Mark
31:11Flemmings Group at Children's Hospital
31:13boss and they were doing some studies
31:15of iron deficiency and pregnancy.
31:17So in pregnant mice that an iron
31:19deficient diet they were able
31:20to induce consistent anemia.
31:22But of course that's a very difficult
31:23experience for a long term experiment
31:25because the mice will eventually give birth,
31:26so it's it's not a practical
31:28way to study this problem,
31:29but it confirmed that this is
31:31a not this played elevation.
31:32We seen the temper 6 knockout might it's not.
31:34It's not something specifically
31:36related to their website and defect.
31:38It's more general phenomena related in
31:40some way to their iron deficiency anemia.
31:42And oh, I wanted to point out, of course,
31:44this is the collaboration with a group
31:46of Diane Kraus 2 proposed excellent postdocs,
31:48a former postdocs,
31:50now Julianna Shaviv Trucchio
31:52and Vanessa Scanlon.
31:53Jade in my lab,
31:54and Larissa in my lab as well.
31:57So as many of you know,
31:59Diane is very interested in hematopoiesis.
32:02This is the classic hematopoiesis tree.
32:05And here we have the megakaryocyte
32:07erythroid progenitor cell.
32:08So this is a cell that can decorate
32:10down the megakaryocyte for the erythroid
32:13lineage and Diane is very interesting
32:16factors that control this fake decision.
32:19So we hypothesized that the megakaryocyte
32:21erythroid progenitor cell from iron
32:24deficient mice would show a bias
32:26towards the mid career site MK lineage.
32:28Diane's lab has an excellent
32:30assay to assess this,
32:31so this is a colony forming assay to
32:33assess the MK erythroid lineage potential
32:35where the megakaryocyte erythroid peak,
32:38which your cells or MP's or
32:40isolated from mouse bone marrow
32:42by facts grown with cytokines.
32:45To promote the growth of
32:47MK and erythroid progeny.
32:49And after several days,
32:50the colonies are fixed and stained
32:52specifically for me to carry site
32:53markers shown here in Green CD
32:5541 and northward markers here.
32:57City 71 the transferring receptor
32:58shown in red so you can get
33:01develop colonies that are MK only
33:04erythroid only or a mix of the two.
33:06And we found that compared to wild
33:09type animals MEP's from temper 6
33:11knockout mice showed an increased
33:14percentage of colonies that
33:16were only forming MK progeny.
33:18So the MEP's from 10 per six knockout
33:21mice were MK biased and in similar
33:24studies we found that there was also
33:27bias in mice would be dietary induced
33:29our inefficiency anemia during pregnancy.
33:34So the next question became,
33:35does the low iron environment itself
33:38promote the mercury site bias of the MEP?
33:41So to address this we moved on to some
33:43bone marrow transplantation experiments.
33:46So in this experiment.
33:47We took temper 6 wild type mice and
33:50iron balance hosts or temper 6 knockout
33:53mice and iron deficient host and they
33:56were transplanted each with wild type.
33:58Were not that bone marrow and I
34:00want to remind you 10 per six is
34:02not expressed in the bone marrow.
34:03So, 12 weeks later,
34:04we looked at the blood parameters
34:06these animals and you can see that whenever
34:08the we had a well typed recipient,
34:10serum iron, blood, hemoglobin and plate
34:12levels were all in the normal range.
34:15The donor genotype for the
34:17marriage didn't matter.
34:18When we put knockout or wall table
34:21mirror into knockout recipient.
34:23They showed the animals showed
34:25cerebral blood parameters consistent
34:26with iron deficiency anemia,
34:28and they showed elevation of platelets.
34:31So we then used the Colony formation
34:34assay to assess the MK erythroid
34:37lineage potential of the MEP's and
34:39you can see that when wildtype
34:41mice were transplanted with wild
34:42type were knockout marrow,
34:44there was no significant difference.
34:45The number of megakaryocyte
34:48colonies produced.
34:49However,
34:49when the recipient was a knockout mouse,
34:53we saw an increase in MK number of
34:56the number of colonies that were
34:59MP's that needed that yielded MK.
35:01Only colonies to say that.
35:04And a similar increase when wild type
35:06mice was put into the knockout host.
35:09So it was really the iron status of
35:11the host was that was determined.
35:12This commitment to the MK lineage
35:15and in a number of further studies
35:16that I'm not going to get into detail
35:18here today we tried to get some
35:20insight into the mechanism by which
35:22this fake decision was occurring.
35:24First,
35:24we tried knocking down expression
35:27of the transfer receptor,
35:28one which mediates iron uptake,
35:30but unfortunately that was that you know,
35:33toxic to the cells,
35:34so that that wasn't a viable.
35:35Approach but in Human MP's we were
35:38able to recapitulate this MK bias by
35:42knocking down transparent receptor 2.
35:44So this is the transformer receptor 2.
35:46I just showed you that in the table
35:48of hemochromatosis genes and it's
35:49not only expressed in liver but
35:50it also seems to play a role in
35:53erythroid cells where it modulates
35:54signaling through equation receptor.
35:56So reducing expression of TFR 2
35:58which is thought to be more of an
36:01iron sensor rather than immediately
36:04requiring uptake seem to modulate.
36:06This MK bias.
36:10For the last few minutes,
36:11I want to talk about a collaboration we've
36:14had with Jackie Fretz and Orthopedics
36:16looking at emerging links between
36:18iron deficiency and the hormone FGF.
36:2023 fibroblast growth factor 23 so.
36:24FGF 23. Is a hormone produced,
36:28thought to produce classically
36:29by osteocytes and bone,
36:31and it plays a critical role in
36:33skeletal health by regulating
36:34the phosphate vitamin D axis,
36:36circulating levels of FGF,
36:3823 rise in patients with chronic
36:40kidney disease, and this FGF.
36:4223 elevation is associated with adverse
36:45cardiovascular outcomes and all cause
36:47mortality in patients both with
36:49and without chronic kidney disease.
36:51Whether or not FGF 23 is a direct mediator.
36:54A biomarker I think is still under debate,
36:57but what was interesting to us
36:58and why others had approached me
37:00about our model is the fact that
37:03iron deficiency had been found to
37:05correlate with FGF 23 elevation
37:07in the circulation of both humans,
37:10healthy humans and mouse models.
37:13And so of course we wanted to turn
37:15to the temperance model again to see
37:16if we could use that as a tool here,
37:18and we found that temper 6 knockout
37:21mice compared to wild type where
37:23heterozygous controlled so it increased
37:25urine phosphate to creatinine ratios as
37:27expected based on the known function
37:30of FGF 23 and increased phosphate excretion.
37:33And looking in their blood,
37:34we saw level increased levels
37:35of the active FGF 23 hormone,
37:37as well as total FGF 23,
37:40probably because it's a different.
37:41A lot of apps say that analyzes.
37:43Both inactive and active forms,
37:45the hormone which undergoes cleavage.
37:49So.
37:49We thought we would be looking at
37:52FGF 20 regulation of the bone.
37:54But when we isolated bone mRNA
37:57from bone cortex,
37:59we actually found that FGF 23
38:01elevation was not increased in
38:03the temper of 6 knockout mice.
38:05And we discovered 2 the better.
38:07We clean the bone,
38:08the clearer this cleaner that she
38:09was all came showing no difference.
38:11So it became very suspicious of
38:12the Bone Arrow itself.
38:13May be the source def.
38:14Chapter 23.
38:15Elevation iron deficiency anemia.
38:17And indeed that's what we saw
38:19when we looked at expression by
38:21qPCR on the bone marrow.
38:25So to get for more of a grip on what the cell
38:28type was that produced was producing FGF,
38:3123 we introduced an FGF 23 EGF reporter,
38:34or little into the upper 6 mouse line,
38:36so this is a well characterized
38:38allele in which the the enhanced
38:40green fluorescence protein, or egfp,
38:43is knocked into the endogenous FGF 23
38:46locus immediately after the start codon.
38:48So this is a reporter allele that
38:52also knocks out FGF 23 transcription
38:55from the same allele.
38:57And mice heterozygous for the supporter,
38:59so the mice that have one functional FGF,
39:0123 little only are known to maintain
39:04normal hot phosphate balance.
39:05So we bred the soul into the
39:06temporal six months line and I won't
39:08show you all the supporting data,
39:09but basically wanted to prove that
39:11temper 6 knockout mice carrying reporter
39:13were still equally iron deficient.
39:15Had equal degrees of anemia and retained
39:17after you have 23 elevation in the plasma,
39:19which they did.
39:21One little a piece of data I'll show
39:23you is inside is temper 6 knockout
39:26mice also have elevated levels of
39:28risk reports and we looked at various
39:30lab parameters trying to see if we
39:32found certain parameters that seemed
39:33to correlate with FGF 23 elevation,
39:35and in fact it was the erythropoietin level.
39:37If we look across mice at different genotypes
39:40that seemed to correlate best with FGF,
39:4223 three levels in circulation,
39:45and just hold that thought for now.
39:48So we had a GFP reporter mouse.
39:50We did flow cytometry.
39:52The total bone marrow to try and
39:54see if population and you can see
39:56that in mice carrying the portfolio
39:58both temper 6 knockout and temper 6
40:00heterozygous with the reporter allele
40:02heterozygous for the reporter level.
40:03Just a very small fraction of
40:05the population with GFP right?
40:07But we did not see these small
40:09fractions right.
40:10Cells in my selected reporter.
40:12This turned out to be less than .02% of
40:15power cells and so Jade was very frustrated.
40:17A bit heartbroken.
40:18She thought these were going to be
40:20incredibly difficult animals to work with.
40:23So we we then took a look at the bone
40:26marrow biopsies in these mice with
40:29Jackie frats and here's confocal of.
40:32Core biopsies after this very brief fixation,
40:34you can see green fluorescent
40:35throughout the Marina patterns,
40:36suggesting localization to the vasculature.
40:40So this led us to think more about our
40:42flow cytometry and using a protocol
40:45to enrich for endothelial cells.
40:47We look specifically at GFP expression
40:49within cells in the endothelial cell gate.
40:52In mice,
40:53the various temper 6 FGF 23 genotypes,
40:57and we found that looking within the
40:59endothelial cell gate mice that carry
41:01the reporter allele showed a subset
41:03of salt with breaking fluorescence,
41:05which was not seen in my slacking
41:08airport earlier.
41:09And the percentage of cells in the
41:11endothelial gate that were GFP bright
41:13was higher in temperate 6 knockouts.
41:15The iron deficiency type compared to
41:17the non iron deficient heterozygous
41:19controlled unit type.
41:25So to gain further expression for
41:28FGF 2323 elevation in expression
41:30in bone marrow and Ophelia cells,
41:32we mind a published data set from the lab.
41:34David David Scadden in the study,
41:37the author use single cell RNA seek of bone
41:39marrow stromal cells from blastic mite sex,
41:426 mice at steady state.
41:44So these are normal mice.
41:46Normal iron balance,
41:47normal phosphate balance,
41:48and in their study they were
41:49able to identify 17 stromal cell
41:51clusters and mining their data.
41:53We found FGF 23.
41:55Expression in the population they
41:58defined as sinusoidal endothelial cells.
42:01Looking more at the individual
42:03single cell data,
42:05you can see that the cells that
42:07express FGF 23 also express,
42:09and Ophelia cell markers,
42:11Pecam 1 and amusin.
42:13And they do not express classic bone markers.
42:17These blue populations of the
42:19osteon lineage populations,
42:20which traditionally are thought to
42:22be the populations producing FGF.
42:2423,
42:24And I'll point out one interesting
42:26observation you found here,
42:28is that erythropoietin express
42:30receptor expression is also detected
42:32in these sinusoidal endothelial cells,
42:34which is why I mentioned the hypo
42:36levels earlier in the correlation with FGF.
42:3823 upregulation.
42:42So to assess expression of the
42:45FGF 23 reporter allele in the
42:47context of tissue architecture
42:48we employed immunohistochemistry,
42:50good old immunohistochemistry with anti GFP
42:52antibody and fixed bone marrow sections.
42:55And you can see that mice carrying
42:57the reporter allele showed stania
42:59sign your little initial cells.
43:02And the staining was not observed in mice
43:04that do not carry the reporter allele.
43:06The staining is more intense and the
43:09iron deficient temper 6 knockout mice
43:11than the heterozygous control mice.
43:13And looking at lower power in one of the
43:15temporal 6 knockouts with the reporter,
43:18you can appreciate that this and
43:19it still still still staining
43:21extends throughout the bone marrow.
43:25We also looked at other organs from
43:27these animals and we detected GFP
43:29expression rare cells of the thymus,
43:31but not in in a variety of other organs,
43:34including liver, spleen,
43:35heart, muscle or kidney.
43:36And I again want to give a special
43:38shout out to Amos Brooks from
43:40Yale Tissue pathology service,
43:41who optimizes IHC staining which
43:43which turned out to be really,
43:46really beautiful. So.
43:49We had shown temper 6 mice expressing
43:53shave evidence of expressing FGF.
43:5623 instinctual endothelial cells.
43:57The catch here is that these
43:59mice have upside and elevation,
44:01so the question is now.
44:04Our bone marrow,
44:05signal hill and ethyl cells is cited FGF 23,
44:07elevation in anemic might have
44:09attacked outside of regulation.
44:11So to test this we use the
44:13FGF 23 Egfp reporter mice,
44:15and this case these mice carried
44:172 well tempered 6 wheels,
44:19so they're not anemic.
44:20They have normal iron status and we
44:22subjected them to large volume colotomy
44:25with intraperitoneal saline volume
44:27replacement to induce acute anemia,
44:29and you can see that 18 hours after flatter
44:32you were successful inducing anemia.
44:34People elevation and elevation of
44:36plasma after you have 23 in the plasma.
44:40And looking at their bone marrow
44:41section at this time point,
44:42you can see upregulation of FGF
44:4423 and sinusoidal of the sorry of
44:46the reporter allele in sinusoidal
44:48endothelial cells of the phlebotomist
44:50mice with the reporter compared to non
44:53phlebotomist mice carrying reporter allele.
44:56So this raises some questions.
44:58Does does FGF 23 have a local
45:00role in the bone marrow?
45:02Is this a protective or
45:05a pathological response?
45:06There is a paper that came out in
45:09blood from a Japanese group last
45:11year that suggested that FGF 23
45:13from a rifter blast is involved
45:15in hematopoietic progenitor cell
45:16mobilization for the bone marrow,
45:18particularly,
45:19this is in the context of induction of G.
45:22CSF.
45:24So exactly how their data fit together
45:26with ours remains to be seen.
45:28I I will say that in in their
45:30paper they do also have expression
45:32of FGF 23 detected in in flow,
45:35sort of populations.
45:36That would include the
45:38sinusoidal endothelial cells,
45:39although they don't specifically
45:41look at that population.
45:43So with that I just wanted to
45:45acknowledge a huge number of people
45:48that made this work possible.
45:50It always takes the village
45:51to do to do research,
45:53starting with the original patients,
45:55their family members,
45:56and referring physicians at Yale.
45:58I've mentioned Jade in my lab who
46:00was a phenomenal PhD student,
46:02Larissa and Outstanding Research assistant.
46:05My mentor at Nancy who got
46:07me started in the field.
46:08I've mentioned collaborations with Mark,
46:10Plumbing Group and Matheny at
46:12Children's Hospital Boston.
46:14Stefano Rebello was instrumental in
46:16providing me with the Palace comic mice.
46:19At Yale,
46:19Jackie's been an excellent collaborator here,
46:21as well as common Bergson,
46:23endocrinology and then my main
46:24collaborators have been the Yale
46:26Cooperative Center of Excellence.
46:27Hematology,
46:28where I've been serving on the
46:30steering committee Dianne,
46:31Vanessa and Juliana,
46:32and her in Diane's lab were absolutely
46:35incredible for our studies.
46:36Steffie Helenus Group has contributed.
46:39Gene Hendrickson's group Pat Gallagher
46:40and also some of the clinical
46:42hematologists who referred me some very
46:45interesting studies where I've been
46:47looking at some exome data on some.
46:49Never look phenotypes as well.
46:51So with that I will stop and take questions.
47:03Very nice. I have several
47:07questions actually first.
47:11So many different families here
47:13in your planning and reaction
47:16relationship with erythrocytes.
47:18Tempress backup cameras 6.
47:22That's also it appears the delivery
47:25of Iron Company backer pitch.
47:28So the Brotherhood islands
47:29in the bone marrow.
47:31Of course he contact with therapist
47:33and order to survive and grow,
47:35so could some of this effect and
47:38ship complete the differentiation
47:40via failure of the Riverside
47:42support by the macrophage.
47:44The other?
47:44Maybe they need to sense
47:45the iron in the back.
47:50I think it's possible, but well,
47:54you're thinking about specifically
47:55upside and effect or just
47:58a low iron effect. I think
47:59you know about your knockout.
48:03They would have trap. They would have.
48:05They should have some relative.
48:06They they may have some relative Miron
48:09trapping in those in bone marrow macrophages,
48:11yeah, but if there is we could that be a.
48:15I remember in style selection.
48:19The grip right precursor is not
48:21in contact with that macro page.
48:27You predicted it would shift it to
48:30a plant. Spontaneous. By default.
48:36Violence and everything.
48:38No, I I will say that the effect of
48:41hepcidin on bone marrow macrophages is
48:43much less well studied than the effect
48:45of macrophages in the spleen or liver.
48:47You know, just people who
48:48were studying mouse models.
48:49It's somewhat easier to to be looking
48:51at these assessments in the screen,
48:52so I don't think it's been quite
48:54as well characterized in mouse
48:56models and have sided regulation.
48:58Just regulation.
49:01And then. Clearly the.
49:05Most of these models show the
49:07iron passing through the cell.
49:09Either it's faster, it's it's
49:12liberated in the auto light lights.
49:16And then it is airborne
49:17and puts it out right?
49:19So we're talking about circulating iron.
49:22To this cell is every cell have breakfast
49:25control its internal iron levels,
49:27and that must be a big important
49:29thing in parasitosis control.
49:31And now it's a bit
49:33right. So so this is this is
49:36another whole side of iron biology.
49:40There's a great review called 2 to Tango,
49:42written by Martina Buffet,
49:43Baller and and several other leaders
49:45in the field where you know upside and
49:47regulations systemic are in regulation
49:48but upside and acts on Fairport.
49:50And of course which is expressed not
49:51only in the cell types I showed you.
49:53In fact, probably on most if
49:55not all cell types in the body.
49:57Cells also regulate iron status through
50:00the iron regulatory protein system,
50:02which stabilizes the messenger
50:03RNA proteins that are involved
50:05in iron transport into the cells.
50:07The transferrin receptors normally
50:09stabilized during iron deficiency
50:11and during iron deficiency.
50:13The translation of proteins involved
50:16in iron exports, such as fair cordon,
50:19is reduced so the cell have means
50:21of compensating throughout.
50:22Tosis is very interesting because the
50:24transfer receptor is not behaving the
50:25way you predict based on that system.
50:27That's something actually asked
50:29during his work talk last week,
50:31I think, yeah.
50:34That that's the big thing now
50:37turn immunity. To regulate that.
50:43Absolutely. Right?
50:50Check chat, there's only one request that
50:53people can hear questions.
50:58Otherwise the ohh OK. The problem.
51:09Yeah. Stick around.
51:14There's a respect box here in
51:16the body that are regulated.
51:18This is level sheets, traumatic.
51:21It's. My death was gross.
51:26So, so I think this area has been explored.
51:31Oh, sorry, so the question is how that
51:34there are areas in the body where his
51:36levels can change dramatically and our iron
51:39levels in those areas regulated as well.
51:42So the individual I know who's probably done
51:44the most work in this area is Yatrik Shah,
51:46Michigan, who's particularly focused
51:47on the role of HIF one and hip two
51:50in the intestinal cells where they
51:53also appear to be modulating iron.
51:55Of taking the cells in addition to
51:57Fairport and so there is a more complex
51:59story than what I told you today but,
52:01but that's definitely an area under
52:03active investigation as well.
52:07I was thinking more like you.
52:12Took my soul.
52:16Of course.
52:21Just in those.
52:24Higher results.
52:27Right?
52:30I think it's it's certainly possible.
52:31I'm not sure if anyone he's really looking.
52:36So you know, truthfully we use an
52:40old-fashioned Perls Prussian blue stain.
52:42There certainly dies that people will
52:44use for for self culture experiments.
52:47Often we use expression
52:49of the transfer receptor.
52:50Messenger RNA is a simple
52:52surrogate because that's so
52:54responsive to the intracellular ion
52:57concentrations of iron concentration.
52:59But I you know that work may
53:01be going on in this field.
53:03I think I tend to gravitate
53:05towards meetings where the focus
53:07is on hematological disorders.
53:08So so that I'm just probably
53:10less familiar with that area now.
53:16Affairs versus fairy these families.
53:20So there are oxidases and reductases around.
53:23Many of these transmembrane.
53:24Passage steps that are also
53:26part of the story. There.
53:28There are many additional players.
53:29These cartoons you know for iron
53:31uptake that I've shipped friend
53:33showed you yeah. I mean no.
53:45OK, thank you so much for
53:47the opportunity to present.
53:52So.
53:59Discovered.