Introduction to Animal Development
Introduction to Animal Development ZOOLOGY 470
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This 16 page Class Notes was uploaded by Mr. Karli Cummings on Thursday September 17, 2015. The Class Notes belongs to ZOOLOGY 470 at University of Wisconsin - Madison taught by Staff in Fall. Since its upload, it has received 45 views. For similar materials see /class/205124/zoology-470-university-of-wisconsin-madison in Animal Science at University of Wisconsin - Madison.
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Date Created: 09/17/15
Zoology 470 7 2009 7 Couree handoute Page 94 Neural Creel TABLE 131 Some derivatives of the neural crest Derivative Cell type or structure derived Radiulabeled Peripheral nervous Neurons including sensory ganglia sympathetic mm systcm PNS and parasympathetic ganglia and plexuses Neuroglial cells Schwann cells Endocrine and Adrenal medulla parllendocrine Calcitonin secreting cells derivatives Carotid body type 1 cells Pigment cells Epidermal pigment cclls Facial cartilage Facial and anterior ventral skull cartilage and and bone bones Connective tissue Corneal endothelium and strolna Tooth papillae Dermis smooth muscle and adipose tissue of skin of head and neck Connective tissue nfsalivary lachrymal thymus Gilbert 85 thyroid and pituitary glands Connective tissue and smooth muscle in arteries of aortic arch origin G 9 8 Alm lnmhsun mallsll on multiple mum i art 6 A Fig 155 Gilbert 86 Fig 155 Amerinr I mtellul hlutol axons EPlLlL l39i lS Neural luhe Derinalnyomme Sclt l lllnlne Nntochnrd Path 2 cells take a dorsolnteral route littween e epidermis and the t lel39lnalnyotonle Rosll39al Neural x Spinal I clk 5t L39ttvtlmlc and Path cellstrawlventrally l k ulwlnilce through this anterior sclerolome Skin broblast provides signal for meianoblast to develop Into a pigment cell Case Case 2 Case 1 Case 2 inicctinn injection results results Steel l l l l h lelanncyles factor m l U Fluorescent Vilexnan Dorsal mm gltngha gt Ventral mot Sehwnnn cells broblast melanoblast SY T P M W39F Mulanl WhlieKlt Mutant Steel ganglia Adrenal r medulla A ll Gilbert 86 Fig 157 No signal tor melanoblast to develop Wolperi 61 al 26 Fig 925 Zoology 470 2009 Courae handout WWWquotquot1191 75W 75 Fig 155 Gilbert Hum 5e Fig 155 g m liMl iiivl39 Ivniulhrlxn39 KrIHHMUXHUIUI n imnnnnc mum ux gin I39Iinnn39m nl0liiim ummm 39hnmuiiiu wlk nrumlh m hum A 39 39 a I L Smpc Q siylum quotWequot 1 sicckvi39s emung w Ellynid imm 39l hvmid mum g fricnid uniLIgc Trachmi ring Gilbert 5e Fig 159 Hindbmin T m idbrail Posterior midbruin Page 95 Anterior Axon Guidance Woipert et a O 7 Longrange cues Shortrange cues 2e Fig 1126 Chemoanraclion Chemorepulsion Contact attraction Contact repulsion 99 Neirins eg Semaphorins eg Cadherin eg Ephrins Zoology 470 7 2009 7 Courae handouta Page 96 A Stages 15 l6 ScnuphurinI Stage 285 T7 LS 2 Cmral plexus Vcnlml ncn c cord Sannrius 13 Control C Reversed Gilbert 85 Fig 1518 Nelrin 2 gradienl Sensory neurons Ii Commissural neuron Ventral lmdy wall Momr epidernmblasls neurons C thrin l gradient Gilbert 85 Fig 1525 mph Nasal Gilbert 85 Fig 1555 A Ephrin New Temporal retina Tectum Nasal retina Zoology 470 7 2009 7 Couree Hamdoute Page 9 Molecular Techniquee in Developmental Biology I Big idea DNA encodes RNA which ane 39 39 39 tmRNAt mRNA in turn are tians1ated into proteins Thus by working with DNA we are working with information required to rnake a protein DNA is a Very quot 39 39 39 39 39 39 39 A P RNA on the other hand is high1y unstab1e and tends to degrade rapid1y and proteins can on1y be studied biochernica11y Mo1ecu1ar bio1ogy is the powerful techno1ogy that a11ows us to work with DNA and RNA and proteins using a number of different techniques DNA molecule I 39 Gene 2 Y9 DNA strand Campbell 55 quotI I l I I II I ll Fiq 175 Q w E tam W mHNA gt v r Cudun t t l l Amino acid Electrophoreeie Big idea Separate proteins or nuc1eic acids DNA RNA by size by driving them through a ge1 made of a po1yrner 1ike polyacrylamide or a ge1 1ike agarose Why do this Since DNA and RNA are rnade of four sirnp1e bui1ding blocks in long chains we can39t tell them apart chemically t39s the number of bases and their 0 en at mattem This technique at 1east a11ows us to distinguish different DNAs or RNAs by sin ifnot by the sequence of the bases they contain Mixture of DNA 3 39 fragments of a differentsizes 7 1 l G Ll E lH E W l8 UJ El 1 2 Longer fragments Power source gt 39GEl Shorter rGIass fragments plates Completed gel Gel electrophoresis of DNA Zoology 470 7 2009 7 Courac lizmdouta Page 10 Nucleic Aclcl Hybridization Big idea Remember that DNA can undergo base pairing with a complemenmry strand of DNA or with a nucleic in the presence of low salt tends to cause complementary sequences to fall apart melt whereas lower temperatures and higher salt solutions allow them to stick together hybridize or anneal Even if the nucleic acid we want to detect is immobilized in a fixed cell or on a membrane we can still make use of this key property In that case the sequence we use to hybridize to the membrane or tissue is called a probe 688 Fuwee 8t 21L 48 Fig iii Hybridization DNA strand DNA Cloring Big idea We want to get DNA in a form that is useful for making lots of it To do this we chop the DNA with a restriction enzyme producing sticky ends and then we ligate the sticky DNA into a vector a piece of bacterial or yeast DNA that has been similarly rended sticky Then we let bacteria do all the work of making this new DNA for us In a Variation of this technique we first take mRNA and make complementary or cDNA n 1 l A l by using an w make L 39 4 4 DNA double stranded and then we clone this DNA Resiriciion enzyme recogniiiun sequences DNA 7 mun MAYquot errant mus Restriction enzyme cuts DNA imo fragments quotto c quot arr Sticky end Addition of DNA from incline source the lragmeuts Stick together V In e W n n c quotM H mm NA Ii Else seals slrandsi v l a r a Recombinant DNA molecule See Campbell 58 Fig 202 Zoology 470 7 2009 7 Couree hamdoute Page ii DNA Librariee Big idea We need a way to study genes outside the animal after all genes are pretty small One way to do this is to make a library ofDNA by taking a big mixture of DNA chopping it up and putting it into a Vector NA from something like a bacterium or a bacterial Virus 7 called a phage that allows us to make lots f different kinds of DNA that can be further studied 2d 35 Foreign genome culupymh Q L remicuon Hecombinanl enzyme Plasml o39 5ee Campbell 5e Fig 206 ecomhlnanlli phage DNA 23 WE Phage Baeterlal clone 4 clone Pusmu nanny Puma nanny Kinda of DNA Used for Libraries Big idea There are two types 1 Genomic libraries made from the DNA in chromosomes and 2 cDNA libraries made from reverse transcribed RNA representing in the correct proportions the m NA that was being made by the cell of interest In some cases we want the chromosomal genomic DNA to make our library This will have all ofthe regulatory elements promoter sites etc and the introns In a Variation ofthis technique we first take mRNA and make complementary or cDNA by using an enzyme called reverse transc 39pmse Then we make this single stranded DNA double stranded and then we clone this DNA cDNA libraries thus faithfully reflect the population omeNAs from the tissue we use to make the library Once the libraries are made they can screened using Various methods to pull out pieces of DNA that are interesting Makng cDNA 3 A 9 RNA lmnscriplasu 3 MM 0 RNA 9 TNT 9 mm 39m 9 DNA i Revnrse lt li39anscl39iplion 9 o 7 o mi gingA o rm 9 rl39 I L N Furvea et al 6e Fig 179 Zoology 470 e 2009 7 Course handouca Fags 12 Folymemee Chaim Keactlori VCR 39 39 39 39 D A 39 the two erands of DNA The key 39 DNA an stands p A n m ur n mpra Aner coollng the whole process can proceed agam and agam RTPCR RT reverse transcrlptase uses PCR to defgct small quanLlLles of CD d f m mRNA Thls allows senlelve detecuon of whether a pamcular mRNA was presentln a sample see 3111381183 Flg 4 12 srsenununrewns DNA changes 9 nun i 1 DNA polymerase 5 dATP polymerasa Targeled dCTP menus m2 Primers A sequen dGTP 339 end oi i 5 17quot each primer Henna 5 73 W CYCLE 2 separate DNA slrsnds E i E 3 539 9 Coollu i 1 allow gtlmm yields 4 J molecules CYCLE 3 yields 3 molecules gtlmm OmLE I plimers lo yields a Primers 2 nannies E E E Campbell 55 p 57l DNA Sequemclmg arid Gemom lce Big idea Usmg a piece of known DNA asa stamng template 39 more DNA complementary to the fll39sL piece of DNA bulwth so we can A Milli r we use pnmers that suck to the DNA than we make a LwlsLl We add 111 two thngs 1 labeled moleonder growmg DNA Lhasa Lh DN lnfallllm hillL L l n ill hv only one base If 44 p T C Gl than we DNA sequence 1 A 4 and can K gene funcLlon between model organisms purely based on sequence slmllanty m 3 Slngleslranded DNA with unknown CTGACTTCGACAA sequence blue serves as a template TGTT Radloactivaly labeled primer 1 DNA polymerase dATP dCTP dTI39P and dGTP 0 Prepare our reaction mlxlums Campbell 55 p 575 ddATP ddCTP ddTTP AddGTP Zoology 470 7 2009 7 Comes hamdoute Page 15 ddATP ddCTP ddTTP ddGTP 8 DNA synthesis m CTGACTTCGACAA 0 Gel electrophoresis 0 Auloradiography dam ddG to detect radloactwe dds bands a a a n Reaction products I I I lt u l a 1 U 39D 39U 390 390 0 0 Longer G c lragmenrs A T eRead c anddeduce G se 7 sequence A otnewstrand c1 oftemplate c A gt T A T Shorter G C fragments c 5 Southern and Northern Blottng Big idea Separate DNA or RNA using gel electrophoresis then blot the nucleic acid onto a membrane that39 s really sticky making a replica of the stuff in the gel Then probe the membrane with a labeled piece of DNA or RNA it might be radioactive or tagged with a molecule that allows it to be detected using a specific chemical reaction etc This allows you to see ifyour mix of RNA or DNA contained the speci c molecule that you39re looking for since hybridization under the light conditions only allows good matches to remain annealed When the blotted nucleic acid is DNA it39s called a Southern blot after its inventor Ed Southern When it39s RNA it39s called a Northern blot to be cute a Northern is shown here Once a blot is probed the blot can be placed against photographic film and exposed to reveal where the probe bound A I B RNA mum u Spucels ml gt gt M 39rmy conmining Support Wick or buffer solution mu paper Add RNA samples to gel and separate Place gel on wet lter paper between two spacers Zoology 470 7 2009 7 CoW66 hamdoute Fag6 W4 7 1 Add radioactive 7 singlvstmndcd probe Weighl Bluuing 11 39 Nilrocellulose r VI 1 lm RNA moves 0 lter bycapillzln acliun gt mm v m I rqmrculiulndmgmrh th mm r mm h Rm sequence m39mlcrml umm RNAqurnml quotrummt mmquot Giib6rt 66 Fig 414 RNA56 Protection Big idea RNAse protection is used in situations when you would use a Northern blot but is more sensitive It uses a radiactiVe RNA to bind to mR such pieces are protected from being digested by RNAse an enzyme that chews up singleistrands of RNA The protected fragments are then run on a gel to separate them by siz RNAse Protection Assay RPA Hibanuclease RNA molecule 5 e EnzymaGC mgeshen 666 Kalthof39f 26 Fig 17m1 8 Zoology 470 2009 Course handouts Page 15 In Situ Hybridization Big idea In situ hybridization is used to detect the time Q location in a tissue of expression of a mRNA by allowing a complementary RNA or DNA probe to bind to the tissue EDNA Fompiemeumry utmlm CImpnund m specn c mRNA 86 limtbccumvspm plv milrd Holesinlhecell dyr when phosphmr is mnovcd made by detergent Cell A mumam g Dignxigenin Alkaline phosphatase 0 label on lln39dine E1 Wash gt IUIlEllI mRNA 1 Add digoxigeninabeled probe 2 Add alkalini plmsphalasercunjugatecl 3 Add hvmical that Incamm a Llark antibody purple dye when phosphale is renmved Llyr culura lhe cell DNA M icroa rray Introduction DNA microarrays colloquially called DNA chips are used to look for differences in the expression of specific genes between two tissues A robot places spots of DNA corresponding to known genes in an ordered array on a glass slide The array can then be probed using uorescent DNA or RNA probes A machine then measures the uorescence intensity at each spot and higher or lower intensities are measured For example microarrays have been used to look for differences in gene expression in cancer cells vs normal cells and ovaIies vs somatic tissues See Gilbert 8e Fig 413 A related technique involves DNA quotmacroarraysquot which contain larger spots see Gilbert 8e Fig 414 1 Isolate mRNA 3 Hybridization Apply the cDNA mixture to a DNA microarray I 4 Rinse off excess cDNA scan mlcronrray for fluorescence Campbell 56 Fig 209 Zoology 470 2009 Courae handouta Page 16 Uemg Anttbodtee Big idea Some antibodies are made by injecting a rabbit or other animal with the antigen of interest This produces polyclonal antibodies ie many different kinds of antibodies specific to the protein of interest are produced However polyclonals are a nonerenewable resource since they rely on the animals immune system to produce antibodies and the animal s blood must be collected Monoclonal antibodies on the other hand are produced by cultured cells and have the additional advantage that they result in a single specific antibody protein that recognizes the antigen We can then use these antibodies to detect proteins and perturb their functions using several methods Folyclonal antibodiea Antibodies and Adhesion 0 Cell membranes Monoclonal antibodiea 3 Immunization 39 with antigen Adhering cells Myeloma cell culture 3 5 35 JV l Isolate lymphocytes Isolated from spleen membrane protein Antibodies K 7 7 r f l l r a 7 Mi to l x b F 000 A 34gt p f k v39 Lymphocytes v lv Myeloma cells Isolated membrane protein Antibodies prevent l i 2 x 10 restores cell adhesion can adhesiun 1 Adapted from Kalho i 15 Fig 2510 Adapted from Life 45 Fig Box 16 Fusion produces hybriuornas quot quotr quot63 tquot quotquot h f 7 i t W 39 J l 7 t r x AL 1 i a y Destroy nonhybrid 39 Assay hybrid cells cells for antibody I Clone hybridomas l l 2 r t g 3 Jg llt ms 45 s Freeze forJ l 5 future use Zoology 470 2009 Comes Hamdouta Page l7 indirect lmmunoetaining Big idea in this technique we detect the presence of a protein by allowing specific antibodies to bind to the protein we39re interested in To see where the first antibodies the primary antibodies a a h e attach a second antibody the secondary antibody to the first one The secondary antibody has been modified so that it possesses a marker usually an e zyme which can catalyze the production ofa colored precipitate or a rescent molecule which can be Visualized with a special microscope ince we can see where the protein is indirectly by these methods they are usually called indirect immunostaining Apia pteoi from Becker et al 55 Guide t Microecopy Fig l5 Allow antlbodlas to blur sntlgon Speclllc nnuhndlu prlmry unllbodlel 29 us anligen ask Y ir Add labeled anllhodlu th bl d primary anllbodles secondary anlltmdles PAGE and Weetem l mmuno Blottng Big idea in this technique we detect the presence of a protein by allowing specific antibodies to bind to the protein we39 interested in after it has been separated by size and transferred to a me bra A mixture of proteins is first separated by size using SD l A 7 hic 39 of proteins by size irrespective oftheircharge Then as in Southern and Northern blotting the gel is transferred to nitrocellulose or nylon Finally the blot is probed using an antibody specific for the protein we wish to e he The result is an immunoblot Western blotting provides information about the size and abundance ofa protein of interest promin wilh lwo A 55 u l subunits A and a slnglo sllbunll ioinud by a disuifido protein plasflc caslng budge sample loaded onto gel by pipette HEATED WITH sos AND MERCAFTOETHANOL nonolively buffer chamellSDS c A molecules anode POLVACRVLAMIDE GELELECTROPHCRESIS I 1 l r B H c H A H slot 039 pulvacrvlnmidu gel 3 a C H adapted from Alberta St at 55 Fig 442 A immunoblot Zoology 470 7 2009 7 Couree Hamdoute Page l8 Alterng Gene F unctlon m Embryoe Introduction Placing a molecule quotat the scene ofthe crimequot during development isn39t enough to show that it actually plays a functional role in a developmental process Ultima ly we want to perform experiments that test the role particular proteins or RNAs play in embryonic development Here again we make use ofDNA and RNA constructs which when combined with the cellular transcriptional and translational machinery ofthe cells of the embryo result in the disruption of the function ofspecific gene products Depending on the model system we are using we use the most appropriate technique from the set listed in the following pages Overexpreeelon of Injected mRNA Big idea If we inject a synthetic mRNA into a cell the protein encoded effects on the developing embryo In the example shown here a f g 4quot 355 mm oocyte is injected with a mRNA encoding a receptor protein Many 71 other twists on this are possible including injecting only a few specific lg 2232 ls in a more advanced emhryo g Abnormal development Adapted from Kaltho is Fig 413 e Antleenee KNAI and Morpholmoe Big idea Remember that RNA can undergo base pairing with a complementary strand of nuclei acid If the complemenary piece of nucleic acid either a short stremh ofDNA or RNA leaves a piece of a partially duplexed mRNA in a singlerstmnded state then an enzyme called RNAse H present in the cells destroys the mRNA with the single stranded stretch The result is the destruction of the endogenous mRNA ie a depletion ofthat RNA The A strand complementary to the strand that produce mRNA is usually called the antisensequot strand hence the name ofthis technique In a much more common and related technique doublerstmnded A is introduced into a cell resulting in L 39 39 39 of the geneofintene t in z RNA interference RNAi RNAi relies on the production of small inhibitory RNAs siRNAs that interfere with transcription In some cellsembryos short hairpin RNAs can be expressed using a promoter that drives expression in a particular tissue resulting in production ofsiRNA only in those tissues expressing the hai in Although the details differ RNA works well in C elegans and other embryos and tissue culture cells Finally translation of RNAs can be inhibited m0 m i i i Morpholinos work well in zeblafish frogs sea urchins and other species Antleemee RNA RNA mm emme KNA Morphollmoe onto mm in a ntraduced into a cell mRNA for Gene x iiiiimmmmmm translation start site WWW l 8 mRNA mutt Sonata in i o 5 chaaaemnta piece by J p by morpholan oligoriocleaide an emvme t against eerie x The pieces guide a mRNA set otoroteiiis to ttie ummmummmummnuumum quotW W Anllscrht RNA Courteay Oic Naturejoumala FUFVSQ 65 Fig 1712 rtie nurmal mRNA is eliooeo min pieces and j destruyed a Zoology 470 e 2009 7 Camera Handouta Page l9 Tramagemlc Animale Big idea in some organisms it is possible to introduce foragn DNA which will be propagated in the germ line i e t he DNA into a iandom spot on a chromosome inkgration in other cases the Orgnlsm can propagate the aim DNA as an however DNA Such animals are called transgenic animals The method used in mice is shown here Mouse Introduce Iransicvmed gt embryos tntu tester mother Femltzea egg GB 1 Test Somallc cells and sperm or Mlcrolnlecl quotansgene O ullspnng tor presenee ut lransgene as Imear DNA mm and breed selecled mice male pronuclaus Transgenlc mouse Making Targeted Mutatloma by Homologoua Recombmatlom Big idea in mice and a few simpler organisms it is possible to replace the normal piece of DNA with an engineered piece of DN rem 39 39 ldetk DN DNA Oftm er m N val K m uLuiiL Thedeslred mammals in some ta urh knocleinl there are four steps to the knockout process Step1 Invuduce the Furezgn DNA mm ES Cell Big irlea Embryonic stein cells or ES cells are cultured cells derived from the inner cell mass of mouse embryos which retain the abili DNA which erest hut ha wt mal slmteglc locations There are two important features of the engineered DNA 39 39 t it the DN antihi ti n m in kinase Zoology 470 2009 Course handouts Targeted insertion by homologous recombination VECTOR TARGET GENE CHROMOSOME WITH IN CHROMOSOME TARGETED INSERTION Page 20 Random insertion VECTOR NONTARGEI GENE CHROMOSOME IN CHROMOSOME WITH FIANDOM INSERTION Figures in this section adapted from Capecchi 1994 56 Am 270552 59 Step 2 Selectfor Homologous Recombinants Big idea Introduced DNA can be inserted randomly or into the homologous site by recombination Cells that have the foreign DNA incorporated at any site will be resistant to neomycin Only if the DNA is inserted at the homologous site will the thymidine kinase sequences be excised and the cell be resistant to gancyclovir This double drug selection selects ES cells that have our DNA inserted via homologous recombination Step 3 Insert Knocked Out ES cells into the Host Embryo i39J GANCICLOVIR v CELLS CARRYING TARGETED MUTATON ELL LADEN WITH NO MEDIUM INSERTION I 1 quot I Q I y CELL CELL WITH TARGETED WITH RANDOM INSERTION INSERTION Big idea Now we have ES cells that carry one knocked outquot copy of our gene ie they are heterozygous for an engineered mutation in our gene of interest We insert these cells into a host embryo In some cases the inserted cells will give rise to gametes carrying e engineered mutation and the result will be that we can breed such mice to eventually obtain homozygous mutant offspring Step 4 Breed Chimeric Mice to Get Homozygotes Big idea The embryos that receive transplanted ES cells are actually a mixture of genetically distinct cells known as a chinera Using mice with different coat colors we can identify chimeric mice who may have some gametes derived from the introduced ES cells By successively breeding these mice and their offspring we can get homozygotes in which both copies of the gene of interest are knocked out Sometimes homozygotes die as embryos in other cases they may survive but have NORMAL TARGETED CHROMOSOME MUTATION 7 W BmcK FEMALE ES CELLS FR 5 sroc 8 BROWN LA Y ALTERED MOUSE STAGE EMBRYO EMBRYO BROWN MOUSE abnormalities The observed phenotypes are of great interest because they tell us about the functions of our gene Zoology 470 7 2009 7 Courae Hamdouta Page 2l MAYqu CmMz HA Making Floxed Allele ne c quotFloxedquot allele It is inserued into the normal sine by the homologous recombination pnocedure we have just discussed The 39 ses of Floxed alleles 1 Conditional knockouts in this approach expression the recombinase is induced by an agent such as doxicyclin or tetia c ne at the desired time This is useful for making knockouts at a particular time in development 2 Tissuespeci c knockouts in this approach the Cre is expressed only in cemin tissues using a tissuerspecific promomr This is useful for knocking out a gene only in cenain cells U 5 in M Uta rite M t tchii K radiation Bi idea Knockouts start with a sequence and hope for a phenotype Muah39ona1 analysis goes in the reverse direction L gene is mutated Moants are a powerfu1 means by which we can in iz g generate animals with specific deye1opmenta1 defects we can phenotypes eVen study lethal defects by mainaining lines of animals that are heterozygous for the genetic defect Now itis possible a in some A case quite suaightforward e to clone the gene which is mutated x a Common genetic sys 39 cl e the fru39t fly Dr phda the nematode worm Caenorhabditi elegam and a relative Eteralyg te El mlyg te ng5 newcomer the zebiafish In flies saturation mutagenesis has been A 6 A 39LI I 1 de e 0 mental pnocesses Such mutagensis screens use a chemical mutagen or Xr or gamma lays to delibeiately geneiate small deleteions or defects in genes some of which produce interesting phenotypes There are two type of mumnts 1 maternal effect quotquotW VW W quot mutants in which a homozygous mother will ear g 3 4 offspring that have an abnormal phenotype no matter Maternal 39 what the genotype of the offspring is and 2 zygotic Effect 9 X 39 mutants in which the offspring must be homozygous for the mutation for them to have the mutant quot 39 quotquot quot mw mm quotquot quot quot Zygotic phenotype X 039 lt2 14 V Adapted from Alberta at al 55 Panel 2H
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