User:Etchevers/Notebook/Conference notes/2009/09/13: Difference between revisions
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*'''[[User:Etchevers|Heather]] 06:42, 17 September 2009 (EDT)''': | *'''[[User:Etchevers|Heather]] 06:42, 17 September 2009 (EDT)''': | ||
=== William Klitz === | === William Klitz on the Ljungan virus and NTDs === | ||
Ljungan (pronounced yungan) virus is a picornavirus. Only transmission to humans is via animals - speculates through human pets coming in contact with carrier rodents. Brown rats in SF are indeed carriers (did test). Speculate may be similar to polio in that 90% exposed are asymptomatic, 9% small fever without consequences, <1% paralysis. | |||
Prevalence of endogenous infection in humans hard to test as people don't seem to develop many antibodies to it, can't test for presence of those Abs. | |||
Drew on [http://www.ncbi.nlm.nih.gov/pubmed/17435445?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum Carmichael/Shaw/Lammer study] on Maternal stressful life events and risks of birth defects in Epidemiology 2007 to see if correlate with cleft, NTD, conotruncal heart defects. | |||
Induced "stress" regimens (see questions) with viral infections. The no stress conditions were 1 mouse/cage, clear water; the "stress" ones were four mice per cage, glucose in water approx to soda levels. Pregnant dams were exposed to virus. Checked for birth defects, stillbirths and diabetes in survivors. No effect of virus in the non-stressful conditions, effect (but no numbers) in the stressful conditions. | |||
Treat with combo of antiserum to PV with [http://www.stanford.edu/group/virus/flavi/2000/pleconar.htm Pleconaril] antiviral agent apparently helped the diabetes type II symptoms. It wasn't clear to me whether the treatment was applied to dams (as it ought to be) or to the surviving babies. | |||
Cycle of pre-eclampsia in Sweden cycles strikingly in phase with the population expansion/reduction cycles of the vole population. Stillbirths also correspond. Checked presence of immunoreactivity to virus in a stillbirth in brain tissue, also in hydrocephalus (was this the same fetus?) - stain is around the blood vessels diffusely. Macrophages + and used Trisomy 21 as "control" - saw IR in 1/20 brains). +++ in a sudden infant death syndrome brain tissue - in the medulla oblongata in the neurons near blood vessels, but also in macrophages in the lung. | |||
Would like new samples for analyses - cf klitz@berkeley.edu and also ljunganvirus.se . | |||
*'''[[User:Etchevers|Heather]] 07:48, 17 September 2009 (EDT)''': | |||
=== Vicky Patterson === | === Vicky Patterson === | ||
Revision as of 04:48, 17 September 2009
 Conference and seminar notes
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Second day of 6th Intl Neural Tube Defects conferenceDania Pastora, NTDs in Nicaragua 2007-8NTD most common CM in Nicaragua, registry started in 2007. NW Ncaragua - Leon and Chinandega For 20K newborns in two years, 800 stillborn, 495 CM's (2.5%) and 43 NTD. No sex bias. NTDs in stillborn = 9/800 (112/10K) and others = 34/19200 (18/10000). SB most frequent, more in males (9 vs 1 for meningocoele (half dead but from another cause - premature for 3 or respiratory distress); 8 vs 11 for myelomeningocoele (4 dead from surgical complications); 2 cases of encephalocoele and 19 anencephales - where the last have no sex bias). 14% mothers took folic acid and only in 2nd trimester. Supplementation not regulated by health authorities. Damn - my battery did not recharge overnight for some reason!! Will take notes on paper and recopy later.
Suzan Carmichael - nutrient intake and risks of NTDs in CaliforniaMosley 2009 - births after folic acid supplementation only study actually found increase in anencephaly. Contribute data to Mosley study also the CA cohort (part of national birth defect study). Oxidative stress nutrients and anencephaly Lutein (caratenoid?) - odds ratio 1 = no association for first quartile (low intake) and last quartile (high) Vit C - low intake OR = 3.2, high intake protective (-0.6) restricted to women w/o supplements - trend was significant. Vit E OR = 1.4, protective 0.5 with wide CI but trend was significant. b-carotene, low intake slightly protective, high intake v protective (0.2) and for iron - high intake increased risk to 2.5 (as a pro-oxidant... but in light of I Zohn's talk wonders what to think now). One-carbon nutrients and anencephaly Methioninem choline, betaine no differences, folate expected and riboflavin low intake increased risk but high intake brought back to normal. This was exaggerated for spina bifida except unexpected methionine. Thiamin and zinc were opposite - esp high zinc intake OR up to 4 for anencephaly, not very significant trends for spina bifida. Glycemic index - higher index expected increased risk but not found, not for other simple sugars - fructose, glucose or sucrose. Is there a ceiling effect of supplementation? *Supplements are weakly if at all protective.* The folate-sensitive NTDs are now being protected in the US at least. Other nutrients in supplements can be helpful in theory but have not seen a stronger association. For example, subsequent to fortification, NTDs did not go down in the CA Central valley region. Question: from Hank: Low choline risk in another study - in blood the levels are related to those of folate. Also, wonder about serine and something else I didn't catch. Comment about higher iron, higher risk.... re Irene's talk? And comment that the protective effect of folates in supplements may be offset by the not-protective effect of additional zinc in those same supplements. Wonder if can check supplement vs non-supplemented mothers particularly wrt iron status as hard to measure. Main sources of iron and zinc coming from meat, wondering about other nutrients eg in animal fats?
Madeleine Brouns on Grlh genesFrom Amsterdam. Not finding most of the 200 NTD mouse genes (usually upon KO) to be defective in humans. Ideal model should be variable phenotypic expn, not lethal, not syndromic. Curly-tail (1950's) and axial defects (1990's) models. Hypomorph of Grhl3 = curly-tail, whereas, Axd not yet cloned? defect at E9-10, curled tail htz in 60% so slightly dominant? and all hmz have E13 SB but only 1/2 at birth. Intrauterine repair? or just reabsorption of some? Possible methionine rescue in diet. Did a tour de genome with 105 markers - look at Chr 15 between CA15-16 to CA15-05 (37.5) containing 6 genes and marker D15MIT250 to discriminate the genetic backgrounds - in 86 embryos from htz crosses, get 100% linkage so confirms region. Genes are Ankrd46, Zfp706 and Grhl2 gene (most likely candidate!!), Ncald, couple others. No mutations in any, check by qPCR but Grhl2 get temporary upregulation between 25-40 somites development in the SB embryos. Grhl3 is reduced expression to get defect. Looking at conserved non-coding sequences. LOD1229, LOD1404, LOD1549 potential regulatory sequences, still are looking for molecular defect. (me: Are there molecular segments aside from Hox genes explaining predilection to NTD at certain sites rather than the older model discussion curvature and mechanical forces, or closure sequence?) For curly-tail, look at Gustavsson BDRA 2008 for criteria - less prolif in hindgut and ntd, too much ventral curvature caudally, inhibits posterior neuropore closure leading to delay = curly tail or failure = SB. Measured ventral curvature of neuropore region from Brook et al Development 1991 to measure an angle between penultimate somite and some other tail feature. Nick Greene has examined ISH expression. Cross between curly-tail and axd do not interact genetically. Expression in hindgut seen by gene trap. Cross of gene trap and axd underway to see if rescue. Grhl3 expression in Axd/Curly-tail, by qRT-PCR no change Up front, Muriel bets that nature may be a retroviral transposable element close to gene - can check by long-range PCR. Pietro Cavalli - clinical approach to assessing NTD recurrence riskdiagnosis + etiology to appropriate risk assessment. Cf book review chapter by Rasmussen SA and Frias JI in 2006: AD and AR conditions with syndromic NTD. In selected populations, association between consanguinity and NTD risk. Chromosomal anomalies cf articles by Hume 1996, Kennedy 1998, Babcook 1995. Recent study using CGH microarray showing region on 13q including ZIC2 and ZIC5 among others (Kirchoff 2009 Am J Med Genet 149A:894). Teratogenic exposure - metabolic pbs (maternal diabetes, Zn deficiency - interesting in light of previous talk on Zn overdose, hyperthermia including fever from viral infections) and drugs of various sorts. Assess NTD recurrence including homocysteinemia or folatemia, venous blood glucose concentration, Hb1Ac. Questions: RJ - what about advanced paternal age? (Cited literature, nothing more to add). Hb1ac only informative early in pregnancy, as when additional blood produced will be diluted down. Reponse can be indicator of glycaemic control rather than oxygen transport - they seemed to be talking at cross-purposes. Janee Gelineau-van Waes on fumonisinFumonisin B1 = FB1 produced by fungal F. verticilliodes. FTY720 drug based on this alkaloid. Both inhibit ceramide synthase to give higher levels of intracellular sphinganine. Sphinganine -> Sa-1-P which is an agonist of S1PR1 (sphingosine-1-phosphate receptor 1). Two mouse lines: LM/Bc which has 1% resorption and 79% exencephaly upon fumonisin exposure, vs SWV mouse which has 15% resorptions and 1% exencephaly under same conditions, dose-response. Placental abnormalities. Uterine natural killer cells (cf Claudia) help remodel arteries in placenta. Get more proliferation of giant tropho cells in LM/Bc strain > FB1 exposure. Increased sphinganine (decreased activity of ceramide synthase) leads to addition of a fatty-acid side chain to make sphingosine. (Eh?) Less S1P (the phosphorylated form of S1) stored in RBC/platelets usually. Release of S1P binds to G-protein-coupled receptors to make chemokine gradients for leukocytes. Sa-1-P is also a ligand for S1P receptors, and this is greatly increased in both strains on fumonisin exposure. Sa-1-P is an agonist not only of S1PR1 but also S1PR3 cf Ohuchi et al., 2008 who showed S1PR1 in the neural folds. S1PR3 is in the surface ectoderm apically. Upon exposure to fumonisin get translocation to nucleus (nuclear envelope?) of cells in the neural tube and increased also in the yolk sac, nucleus of giant trophoblasts. 1-deoxy-sphinganine (aka spisulosine) from a pink-necked clam, causes cell cycle arrest, under development for prostate cancer treatment. ENStemA cells treated with fumonisin increase intracellular levels of Sa-1-P. Microarray analyses: Males crossed to treated=with-FB1-or-FTY720 / or not females, different lines LM/Bc or SWV. Took E9.5 cranial neural tissue. Saw reduction in Protein phosphatase 2A (B56 subunit), CerS1, S1P3R, and also misregulation (not known which direction) of ciliary and Wnt-signalling pathway genes. FB1 or 1 deoxy-sphinganine block PP2A normally decrese in placenta upon cell cycle exit. The Xenopus knockdown model has NTDs, as it's involved in Wnt signaling. Recent work from April 2009 on kidney cells show loss of cilia upon fumonisin exposure. Metabolism when serine levels go down, use alanine to make 1-deoxy-sphinganine.
William Klitz on the Ljungan virus and NTDsLjungan (pronounced yungan) virus is a picornavirus. Only transmission to humans is via animals - speculates through human pets coming in contact with carrier rodents. Brown rats in SF are indeed carriers (did test). Speculate may be similar to polio in that 90% exposed are asymptomatic, 9% small fever without consequences, <1% paralysis. Prevalence of endogenous infection in humans hard to test as people don't seem to develop many antibodies to it, can't test for presence of those Abs. Drew on Carmichael/Shaw/Lammer study on Maternal stressful life events and risks of birth defects in Epidemiology 2007 to see if correlate with cleft, NTD, conotruncal heart defects. Induced "stress" regimens (see questions) with viral infections. The no stress conditions were 1 mouse/cage, clear water; the "stress" ones were four mice per cage, glucose in water approx to soda levels. Pregnant dams were exposed to virus. Checked for birth defects, stillbirths and diabetes in survivors. No effect of virus in the non-stressful conditions, effect (but no numbers) in the stressful conditions. Treat with combo of antiserum to PV with Pleconaril antiviral agent apparently helped the diabetes type II symptoms. It wasn't clear to me whether the treatment was applied to dams (as it ought to be) or to the surviving babies. Cycle of pre-eclampsia in Sweden cycles strikingly in phase with the population expansion/reduction cycles of the vole population. Stillbirths also correspond. Checked presence of immunoreactivity to virus in a stillbirth in brain tissue, also in hydrocephalus (was this the same fetus?) - stain is around the blood vessels diffusely. Macrophages + and used Trisomy 21 as "control" - saw IR in 1/20 brains). +++ in a sudden infant death syndrome brain tissue - in the medulla oblongata in the neurons near blood vessels, but also in macrophages in the lung. Would like new samples for analyses - cf klitz@berkeley.edu and also ljunganvirus.se .
Vicky PattersonMore characterization of tubby-like protein 3 (tulp3). Mass spec on pull-down with His-tagged protein. Underway. Describes the optimizations and purification of the protein. No increased apoptosis, some increase prolif in hindbrain. Less Sox10+ in cranial nerves - wonders about impact on CNCC. Had asked for good markers, I had suggested AP2 or Wnt1. Subcontracted Y2H to hybrigenics with mouse embryonic brain. 11 putative interaction partners. trim71 (expn ubiquitous but let7 miRNA targets it to restrict, nup155, rgnef... etc. Not expressed in NT, lots of mesodermal mesenchyme in chick. Rgnef is reportedly ubiquitous but see good expn in floorplate at E8.5 and E9.5 as well as in somite or in dorsal roots cf sections. interacts iwth RhoA. Much stronger in cranial region, weaker caudally. Will validate with Co-IP. Muriel asked about work on cranial neural crest and whether migration can cause exencephaly - any mutants where shown to be the case? Re-asks Andy. Says that there are mutants where both things go wrong but no proven causality. Expansion of Shh expn is only in caudal region, starting just anterior to hindlimb bud, extending to 2ndary neurulation region. Rab23 does this too. Claudia noticed that more prolif in mesoderm as well as in NT, answer only in cranial not caudal areas. Delphine PsychoyosMarijuana use and increased risk for miscarriage, anencephaly, other neurodevelopmental effects. Use chick embryo to test. delta9-THC has increased nearly 10x over last ten years, dependent on certain strains of marijuana. THC also lab-produced. van Gelder et al. 2009 found marijuana associated with anencephaly in first month of pregnancy. Although some misclassification of drug use, still pretty evocative. Mid-gestation foetuses also vulnerable eg DOPA2R mRNA decreased in mesocorticolimbic system (Wang 2004). Bypass maternal metabolism to test analog of THC - water soluble, low concentrations, applied in New cultures for 6-21h, then ISH. treatment at pre-somitic stages, SOX2+ neural plate stays flat and no MHP, foreshortened Delta1+ presomitic mesoderm. Later, little extension of neural tube. The Otx2+ forebrain is foreshortened as well (O2545 = drug) - neural tube is flattened. Zebrafish studies by other authors get curvy tails. Examined presence of endocannabinoid system in early embryo, presence of CB1R and DAGL/MAGL metabolism enzymes. RT-PCR on microdissected Hensen's node, CB1R and synthesis/degradation of endocannibinoids are all transcribed. Everything is transcribed at all stages and all ages examined. CB1R via antibody - somites AND neural tube, Jo Begbie had found transcribed in the somites only. CB1R by Western blot in HH4, HH10. Would have liked to see some negative controls on this.
Sandra de Castro Lmnb1 modifier geneLooked for curly-tail targets using 2D gel comparing Ct (Grhl3) and wt strains - proteomics to ID via mass spec Lmnb1. Changes in sequence of lamin B1 - are they polymorphisms? checked various inbred strains (lots of them!). GAG repeats (8 or 9) Splotch embryo on CBA/101 background, as well as other prone strains (CBA does not spontaneously develop spina bifida but is an at-risk background) have the 8 GAG repeats rather than 9. Plays a role in nuclear shape and assembly of nuclear pores, modulates transcriptional regulation and directly influences chromatin structure. test by making ct substrains with combinations of lmnb1 polymorphism and grhl3 mutation. Collected hundreds of embryos with the three genotypes. Percentages of embryos with each phenotype curly tail and/or SB. the SB frequency is significantly reduced in the 9E variant re: the 8E variant background of lamin B1. 2D protein gels showing variations of the spot corresponding to laminB1 re: the background - the glutamic acid deletion causes the shift on the gel. So lmnb1 polymorphism can increase risk of NTD. Function of lamin B1? Effect on cell cycle? Sequencing samples of human spina bifidas for polymorphisms. Human gene also has 8 glutamines. Other spots may have changed as well. ANy known disease? Overexpression causes late-onset leukodystrophy. Lamins A and C also cause human disease. Possible modifier for the Sp phenotype?
Ciprian Marius Bosoi on Prickle 1Works with Jacques Michaud as department head or something like that. PCP discussion. ALso used Kibar et al., schematic. Glad I redrew it. ;-) Dishevelled has PET and LIM domains - so not just interact with Wnt pathway but potentially ISL1 or Ldbs? 810 nonsyndromic patients SB from Genova and 399 US patients from children's memorial hospital in Chicago mostly myelomeningocoele. Lots of controls. 10 missense rare variants. 9 absent from all controls and one was recurrent in six patients. pAsn81His, pILe69Thr, pThr275Met, pArg682Cys, pSer739Phe - all evolutionarily very conserved, in functional modules. Could have tried Polyphen as additional argument? Checking interactions of lim/pet domains with dishevelled and vangl2/diversin interactions with diego domain, of prickle1. Mutagenesis in mouse? Works in Zoha Kibar's group. Pk1 mouse mutants recent paper - hmz died very early and htz survive. Amber Marean and Line2, Line3 and Shroom3 miceLine2 - open face and open body wall - 1Mb region in which to find a gene. Line3 most penetrant phenotype = polydactyly but they can be microphthalmic but also exencephaly 20-40%. Looking at long-term exposure to folic acid supplementation by having multiple generations be exposed to high FA diet based on crooked-tail response (10 mg/kg) and then "normal" which is 2 mg/kg. Cross first gen to get 2nd gen, and look for response due to FA supplementation. Line2/Shroom3 does not seem to decrease phenotype of NTD. But for Line2, rescues the hmz embryo loss/reabsorption. Also, oddly, the htz displayed exencephaly on the "high FA" diet. Shroom3 is losing even more hmz embryos on the "high FA" diet. FA increases incidence of exencephaly from 20 to 60%, polydactyly from 80% to 100%, loss of 1/2 expected hmz embryos. Is multigenerational exposure such a good thing, then? Gene-environment interaction going on. Questions revolved around translating data to humans - mice eat more food per body weight and also don't want to scare off/take out of context relative to fortified flour, etc.
Jason Gray on crooked tail (Betsy [ME] Ross lab)Lrp6 G494D mutation. Co-binds with Wnt to Fz to activate beta-catenin canonical pathway. Dkk1 fails to antagonize the Lrp6-Cd mutation. Lrp6-null embryos with NTDs but different phenotype - highly variable and usually limb defects with split hand, axis body truncation, sometimes SB or some exencephaly. Did breeding scheme like what Amber showed earlier. Got fewer SB/exencaphaly on high diet but in fact not getting a Mendelian distribution of embryos to get Lrp6 nulls. 70% reduction, though average litter size was not changed (7-9 embryos each time). Harvest usually at E13. See 10% resorptions, still not Mendelian ratio. 90% viable on low-folate background, 38% viable in high diet, many started resorption during neurulation. Not seen in WT background as far as resorption rate - effect of Lrp6 gene. tissue culture DMEM has 4 ug/ml folates. Trest with Wnt3a - folate deficient cells inhibit Wnt signaling but also too high doses - 10, 50, 100 ug/mL. Growth-response curve. GOF allele can be rescued by FA - LOF allele can be exacerbated by FA supplementation. Comment - may inhibit rather than supplement the folic acid pathway by these high doses of folic acid provision via feedback mechanisms. Can LRP6 act as switch from canonical into non-canonical pathway? Failure of DLHP not apically constricting, loss of F-actin so could be impacting planar cell polarity but no direct implication. Muriel - absorption 18% on C3H rather high, expect 10% - wonder if 2 ppm is enough for sufficient implantation? Cells have adapted best to this high non physiological doses. Can be reproduced in MEF? (but they are hard to transfect). Pass out back into physiological levels of folate in media is possible. Cf. Hank's objections.
Heather Hamner : findings from National Health And Nutrition Examination Study 2003-6Acculturation in Mexican-American women. Take a look at the abstract for details (last one of the day in the order of the abstract book).
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