User:Etchevers/Notebook/Conference notes/2009/09/12: Difference between revisions
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Histone me inhibitor on lysine = chaetocin, HDAC inhibitor TSA. No effects in undiff condition, but addition instead of the antimycin treatment will inhibit Pax3 expression (relative expn, using qPCR). | Histone me inhibitor on lysine = chaetocin, HDAC inhibitor TSA. No effects in undiff condition, but addition instead of the antimycin treatment will inhibit Pax3 expression (relative expn, using qPCR). | ||
''Questions:'' | ''Questions:'' | ||
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Role for inositol supplementation cf diabetes? Alleviation reported in the rat in vitro… Fatty acids work as antioxidants. Or substrate for something playing a signaling role? | Role for inositol supplementation cf diabetes? Alleviation reported in the rat in vitro… Fatty acids work as antioxidants. Or substrate for something playing a signaling role? | ||
(I wondered about available plasma vitamin A or retinoid levels in diabetic women - cf [http://www.ncbi.nlm.nih.gov/pubmed/18437353 that study].) | |||
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Question: is group getting supplements only from grains sufficiently protected against vitamin A deficiencies? Answer: don’t really know. | Question: is group getting supplements only from grains sufficiently protected against vitamin A deficiencies? Answer: don’t really know. | ||
*'''[[User:Etchevers|Heather]] 13:55, 12 September 2009 (EDT)''': | *'''[[User:Etchevers|Heather]] 13:55, 12 September 2009 (EDT)''': |
Revision as of 12:02, 12 September 2009
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First day of 6th Intl Neural Tube Defects conferenceI arrived super late the first morning thanks to an epic 34-hour journey during which I slept 6 in a bed. First talk where I took notes: 6th Intl NTD Conference: first day = September 12, 2009 • Claudia Kappen – diabetic embryopathyRecent publication – effect on Wnt pathway genes in the induced diabetes model (injection of mice with streptozotocin, down insulin, blood sugar up, mate the dams at day 15. Get 10-15% NTDs at E10.5 and beyond. Genes mostly downreg in the embryo proper, also looked at and mostly reported the results in the placenta. Prevention of diabetic NTDs with folic acid (somewhat controversial). Breeder diet before mating dams >= 6 weeks. Timing of gene expression changes in induced diabetes model is different depending on Chow or Breeder diets (differ by 10% protein/fat, less folic acid in latter, same carbs). On Breeder-fed FPB mice, get more NTDs in diabetes-exposed (22% vs 6.6% on Chow diet) embryos. But revealed at the later stages in development, not seen by looking only at E10.5, also look at E12.5, E15.5, E18.5 (increasing incidence). Macrosomia in diabetic humans – different in mouse. How much related to the maternal obesity to start? Rarely observed in rodent models, but the diabetes is very severe. Questions: Genetic reasons for dietary effects of Chow vs Breeder in other strains of mice 4 misregulated imprinted genes (3 down, 1 up in diabetes) and 3 regulators of imprinting are misregulated. Since 10.5 is > neural tube closure (can ascertain if NTD then) then how relevant? Maternal diabetes changes in the affected embryos? Variance is as great as in the non-affected exposed embryos. • Mary Loeken - ChIP on Pax3 promoterDiabetes much reduces Pax3 – at E10.5 the exencephaly looks like the Splotch phenotype. Less expn at E8.5. Not all the embryos develop malformations. Stochastic NTD occurrence re: exposure to hyperglycemia (glucose necessary and sufficient to inhibit Pax3 by inducing oxidative stress) during a critical window. Drug induces oxidative stress: antimycin A; antioxidants reduce hyperglycemia. Using ES cells to plate and study molecular regulation. (Experiments described in D3 cells derived from 129 Sv strain.) Transfer ES cells to Petri dish and RA stimulation to form embryoid bodies. Select for nestin + precursors on ITSF + medium, get neuronal precursor-looking cells on the newly adherent plastic. Pax3 induced on differentiation, but abrogated with antomycin A, some other gene expression is maintained including nestin. Rather than high glucose: the D3 cells are cultured in a high glucose DMEM anyhow and have lost glucose response. Differentiating cells with or without ox stress. Perform ChIP. PCR Pax3 5’ elements. “Pan-H3” antibody, H3K27me3 (decreased expn), H3K4me3, H3K9ac (increased expn). 1.6kb upstream – strategy = primers amplifying overlapping 200bp amplicons, overlap by 100bp. 1.6kb directs Pax3 expn in NT aside from a part of the head and NCC. Doesn’t control expn in somites. -180 and -400 seems to be concentration of sites. What other chromatin modifications provide access? Get changes at best 1.5-2x, but error bars are very reproducible (relative IPm though to what?). Change at -1000 to -1200 on differentiation. H3K27me3 – no real changes but on a log scale and overall much noisier. H3K4me4, get changes over 3 conditions (before diffn, after selection/readhesion, and after but with antimycin A). Eg -200 to -400 v.low to start (0.01, then 1 for the after, and back down for antimycin treatment). Histone me inhibitor on lysine = chaetocin, HDAC inhibitor TSA. No effects in undiff condition, but addition instead of the antimycin treatment will inhibit Pax3 expression (relative expn, using qPCR).
Folic acid deficiency by up homocysteine, ionizing radiation, other causes of oxidative stress. Specific kinds of free radicals are responsible - increased superoxide production eg. What happens in Pax3 htz? Could not induce C57Bl/6 background maternal diabetes effect NTD even in the Pax3 – and this is a dominant effect. But Splotch on another background, maybe – difference due to glucose transporters? Would like to do ChIP-seq perhaps. Role for inositol supplementation cf diabetes? Alleviation reported in the rat in vitro… Fatty acids work as antioxidants. Or substrate for something playing a signaling role? (I wondered about available plasma vitamin A or retinoid levels in diabetic women - cf that study.)
• Paul (Kit Sing) AuGlucose metabolism genes associated with spina bifida risk or AD/HD in SB patients. 22% white US women obese, 24% men; Canada 25%; black and Hispanic populations are around 30%. Leptin and glucose transporter (glut1/2/3) genes SNPs associated with increased BMI. Cf. Davidson 2008 – SNPs and SB. How well do lymphocytes from patients take up glucose? Isolate from patients and mother, test under 3 conditions – normal, hi glucose, sucrose conditions, do they take up, according to GLUT1 haplotype? (or HK1, or LEPR) genotypes: the significant results are from the LEPR genotypes. Subdivide populations into these three subpopulations. Some SNPs have sig differences, using TDT instead. 140 SNPs analyzed using RC-TDT: BRCA1, GLUT1,/3/4/8, HK1, LEPR, INSR, LEP, SOD1/2, TP53. Plot –log p and get significant results for many SNPs in GLUT1 in particular rs3339682 reproduce previous findings from other groups as associated with SB in patients. Linkage disequilibrium of 10 sign GLUT1 SNPs comparing Hispanic vs Caucasian populations, but though seem different he thinks they are not actually… did not quite catch this.
• Mary E. CogswellFolic acid consumption large study including diet (enriched cereal grain products w/o ready to eat cereals), ECGP+cereals, ECGP+cereals+supplements. Used PC-SIDE software for intake distributions estimation, also accounted for race, ethnicity, sex, BMI, day of week as weekend diet can differ from weekdays. Calculate nutrient concentrations through a whole series of stat methods I am not qualified to assess but sound good. Complex sampling design. Usual total folic acid daily intake in 8,258 US adults. Median = 288 ug per day (160-462) and 2.7% at 95CI consumed >1mg. ECGP only 42% of adults, median = 138; ECGP+RTE cereals 274 ug, none exceeded tolerable upper limit. ECGP+supplements, 479 ug, 5.5% exceeded UL, 635, 9.4% exceeded UL. Consistent across all race, ethnicity, etc. Adults aged 65 and over 12.8% exceeded the upper limit and had ECGP, supplements and breakfast cereals. 11% adults up to 200 ug/day from supplements. Less than 1% exceeded UL. >400 ug/day, 48% of these exceeded the UL. 200-400 ug/day, again less than 1% exceeded UL % US adults with high serum folate and low B12 concentrations inverse relationship where up to 30% of ECGP+RTE cereals+supplements had >20ng/mL but decline with other sources of folic acid (from vitamin B12?). I didn’t get it – went too fast. But it keeps homocysteine down. Supplements for folic acid also have B12 reducing risk for B12 deficiency. Despite contributions from diet, RTE cereals and supplements <400 ug/day, 94% US adults won’t go over the recommended tolerable upper limit of 1 mg/day.
• Sarah Tinker, CDC also.Women of childbearing age but non pregnant in the cohort previously described. USDA and public health service recommendations – 400 ug/day if trying to get pregnant. Some recommendations up to 800 ug/day (USPSTF where TF is task force). Women with obesity or diabetes don’t particularly increase their folic acid intake and don’t make particular population-visible efforts to take supplements – conclusion, they should, because no women of childbearing age sub-populations are really able to get to the recommendations on diet alone. Questions: Tough with non-supplemented diet to get folate levels. Before supplements between 100 and 10000 years ago, was there a selective impact on the species? Was there another dietary source or were all those particular babies lost? Answer (of course): I don’t know! RCT’s were only done on synthetic folic acid. Other members of the audience chipped in: But although we think we are sophisticated in our eating habits, earlier populations may have actually eaten more appropriate food to prevent certain vitamin deficiencies, when the food itself was available. “Paleolithic diet” currently in fashion – yet would have it been up to 400 ug/day?
• Jenny Murdoch (Oxford) – exencephaly and SBHitchhiker mutant – modulator of the Shh pathway. Neural tube expansion. Sections are completely open book but sometimes large tissue oedema and luminal expansion, also not a proper roofplate. Dorsal view sometimes open SB but also splayed vertebral splay and thin skin covering that (SB occulta)? This looks like patterning DV defects. Vertebral defects: they are splayed. Mutant is tulp3, a splice site mutation. In WM of E10.5 Shh expression normal in head then much more in the caudal region of the embryo. In section, expands Shh expression all the way up to the alar plate. Get dramatic ventralization - Nkx2.2 and Pax6 looks pretty normal though says dorsalized, however, Msx really does look more restricted dorsally. Patched1 is overexpressed in caudal end of E9.5 mutants - increased activation of pathway by tulp3 at a point where Shh expression itself is still pretty normal. Loss of Gli3 repressor, activation of Gli1/2. Position tulp3 in there by genetic interactions - cross hhkr mice to Shh and Smo mutants. Souble hmz rescues the gross phenotypes especially exencephaly instead of holoprosencephaly, but no rescue of heart defect in Smo cross (hhkr acts downstream of Smo as well as of Shh). Rescue however does not compensate all head problems - synophthalmic rather than cyclopic. tulp3/gli2 double mutant looks more like gli2, so genetically upstream. Gli3 processing not altered. Gli3 mutant only subtle DV patterning problems. Somehow normal role of tulp3 to prevent activation of the Gli activators. Crossed to mutant lacking cilia - kif3a - need cilia to get the tulp3 ko phenotype. Ultrastructure from limb buds E11.5 embryos WT or mutants - same length, normal shape in hhkr mutants. Immunostain with polaris and still apparently normal cilia. Tulp3 expressed in the tip of the cilia! that is lost from the null allele - but it is also seen in the cell nucleus - wonder if it translocates. Want to try real time imaging for subcellular localization. Putative TF - nuclear localization, has a transcriptional activation domain in hybrid assay, and a DNA binding tubby domain. However, no evidence for role as a TF on components of the Shh pathway. Microarray analyses - caudal end of E9.5 - nothing but some DV markers changed. Protein ChIP with antibody but found no specific binding sites (admittedly not the best assay, but supports other data). Intracellular trafficking? PI linkage attaches to membrane. Doing a yeast two-hybrid assay (cf talk tomorrow from student) on a mouse embryonic brain library. (Vicki Patterson). Questions: What upregulates Shh in the mutant, then? But Shh pathway is activated and leads to increased expn in neural tube, not sure if perhaps Gli2 not properly repressed, and b/c activator more active than normal may lead to more ventralization. Expression pattern of Tulp3? Ubiquitous expression - no AP difference. Have not looked at protein level, though tried Westerns - why not immunohistochemistry? Vertebral phenotype. Somite patterning normal or not? (will look). If embryo develops further, double hmz eye malformations. Any notochord duplications? and any other anomalies? Only let them develop to E12.5. Narrow head and close-set eyes. Also limb defects. Double hmz forelimbs have two digits, hindlimbs have four. Incomplete activation of Shh by Tulp3. Also don't rescue floorplate marker expression. Think to mention to her Yuji and Anne-Helene's work in which Shh can inhibit Msx expression and thereby the formation of the spinous process. What is interesting is why the regulation is segment-specific along the length of the neural tube - why only caudal?
• Adriane Griffin- National Council on Folic AcidSpina bifida association - coalition building for change. 62 members of coalition - nonprofits, national organizations, etc. How get messages to women? Current shift from web-based portals to hand-held devices, as an example. Hooks for messages - need to present it eight times for it to register with the targeted audience. This is more about communication efforts and how to go about it, and not the right audience. But it's nice that someone who knows wheat they are doing in communication is behind the cause of making sure research results reach the women who could benefit from it.
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