Transport of fatty acids within the cytosol of adipocytes and their subsequent assimilation into lipid droplets has been thoroughly investigated; however, the mechanism by which fatty acids are transported across the plasma membrane from the extracellular environment remains unclear. Since triacylglycerol-rich lipoproteins represent an abundant source of fatty acids for adipocyte utilization, we have investigated the expression levels of cell surface lipoprotein receptors and their functional contributions toward intracellular lipid accumulation; these include very low density lipoprotein receptor (VLDL-R), low density lipoprotein receptor-related protein (LRP), and heparan sulfate proteoglycans (HSPG).We found that expression of these three lipoprotein receptors increased 5-fold, 2-fold, and 2.5-fold, respectively, during adipocyte differentiation. The major proteoglycans expressed by mature adipocytes are of high molecular weight (>500 kD) and contain both heparan and chondroitin sulfate moieties. Using ligand binding antagonists, we observed that HSPG, rather than VLDL-R or LRP, play a primary role in the uptake of DiI-lableled apoE-VLDL by mature adipocytes. In addition, inhibitors of HSPG maturation resulted in a significant reduction (>85%) in intracellular lipid accumulation.These results suggest that cell surface HSPG is required for fatty acid transport across the plasma membrane of adipocytes. The adipocyte plays a central role in overall metabolic regulation serving as a storage depot for fatty acids and as an endocrine cell to regulate energy utilization and feeding behavior ,2. The mExtracellular fatty acids that are available for adipocyte uptake are either 1) associated with circulating albumin, 2) hydrolyzed from triacylglycerol-rich lipoprotein particles by lipoprotein lipase, or 3) in the form of VLDL particles which can be directly internalized by adipocyte lipoprotein receptors. In the circulation, VLDL represents the major source of fatty acids for peripheral tissues in the form of triacylglycerols and provides a concentrated source of esterified fatty acids. It is interesting that in light of the well studied processes of cytosolic transport and assimilation of free fatty acids into triacylglycerol-rich storage droplets, the mechanism of transport of fatty acids across the adipocyte plasma membrane remains controversial. Two mechanisms, which are not mutually exclusive, have been proposed: one involves passive diffusion across the plasma membrane ,7, the oIn vivo studies corroborate these observations as uptake of long-chain fatty acids is impaired in CD36 knock-out mice [Several cell surface proteins are expressed by adipocytes which potentially contribute to receptor-mediated uptake of extracellular fatty acids; these include CD36, fatty acid transport protein-1 (FATP1), very low density lipoprotein receptor (VLDL-R), low density lipoprotein receptor-related protein (LRP), and heparan sulfate proteoglycans (HSPG). CD36 is a cell surface glycoprotein that binds long-chain fatty acids with high affinity and demonstrates a subcellular distribution that is consistent with a role in fatty acid transport across the plasma membrane -23. Follout mice  or when out mice . Althougout mice . FATP1 iout mice . In adipout mice . Similarout mice ,28.Studies using a VLDL-R knock-out animal model have suggested a role for this receptor in the accumulation of fatty acids by adipose tissue; mice lacking VLDL-R demonstrate only modest weight gain and a reduction in adipose tissue mass when placed on a high-fat diet . VLDL-R To better understand the process of fatty acid transport across the plasma membrane of adipocytes, we have examined the expression levels and functional contributions of lipoprotein receptors, VLDL-R, LRP and HSPG toward intracellular lipid accumulation. Our findings suggest that cell surface HSPG play an essential role in fatty acid uptake and intracellular lipid accumulation in adipocytes.in vitro model for adipocyte differentiation [Since members of the LDL receptor family are known to play a predominant role in the uptake of lipoproteins, we chose to examine the protein expression levels of VLDL-R and LRP during adipocyte differentiation using 3T3-L1 cells , which antiation ,43. 3T3-ntiation  and LRP ntiation .125I-RAP at 4°C  and p-nitrophenyl-β-D-xylopyranoside (pNP-Xyl) are reagents that can serve as alternative acceptors within cells for heparan sulfate moieties and are thus able to compete for heparan sulfate chain addition to proteoglycan core proteins ,52. The To determine if HSPG are involved in intracellular lipid accumulation by mature adipocytes, we assessed cytosolic lipid droplet formation in induced 3T3-L1 cells following treatment with either 4-MUmb or pNP-Xyl. Again 3T3-L1 pre-adipocytes were incubated with 4-MUmb or pNP-Xyl concurrently with the addition of differentiation reagents and intracellular lipid accumulation was assessed by Oil Red O staining. Upon Oil Red staining, untreated mature adipocytes demonstrated large intracellular droplets; the characteristic morphologic feature of cytosolic lipid accumulation Fig. . TreatmeIntracellular lipid accumulation is a hallmark event of adipocyte development and the major factor in adipocyte hypertrophy. This prompted us to investigate the molecular mechanism by which adipocytes take up extracellular lipid components. In the present study, we provide new information on the expression levels and functional activities of certain cell surface receptors in mature adipocytes that are known to play primary roles in lipoprotein processing and clearance. These receptors have been extensively studied in liver, the major organ for dietary lipoprotein clearance , howeverThese findings offer a novel observation that cell surface HSPG appear to be essential for lipid accumulation and lipid droplet formation in adipocytes. However, this observation also raises the question as to the exact role of HSPG in the transport of fatty acids across the adipocyte plasma membrane. Based on the results presented in this study and those reported elsewhere, we have drawn a testable model to define how HSPG contribute to intracellular lipid accumulation by adipocytes Fig. . HSPG arThe identity of this HSPG is currently unknown; however, its large relative molecular mass and composition containing both heparan and chondroitin sulfates are consistent with the structural properties of the syndecan ,61 and pThe availability of circulating triacylglycerol-rich lipoproteins for adipocyte utilization and storage relies on transport of these particles across the endothelial barrier. Transcytosis of albumin across endothelium has been shown to occur by a vesicular transport pathway -70 and iHow HSPG might coordinate its activity with CD36 or FATP1 for fatty acid uptake or possibly with members of the LDL receptor family for assisted internalization is under current investigation. Presently, our results provide novel findings indicating that cell surface HSPG activity is necessary for lipid accumulation by adipocytes. It is anticipated that these observations will aid in our understanding of the complex mechanism leading to adipose hypertrophy and obesity, and provide a novel avenue to explore for targeted reduction of intracellular lipid accumulation35SO4 were purchased from MP Biomedicals (Irvine, CA). p-nitrophenyl-β-D-xylopyranoside and 4-methylumbelliferyl-β-D-xylopyranoside were from Calbiochem (La Jolla, CA). Heparin, heparinase I, chondroitinase ABC, p-nitrophenylbutyrate, lipoprotein lipase, and Oil Red O were purchased from Sigma-Aldrich. Optiprep was from Greiner Bio-One (Longwood, FL). DiI (1,1'-dictadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) was purchased from Molecular Probes (Eugene, OR). Apolipoprotein E was obtained from Calbiochem. RAP-GST fusion protein was purified as previously described [Anti-LRP polyclonal antibody was raised against an 18 amino acid peptide from the cytoplasmic tail of human LRP  and antiescribed . Tissue 2 and passaged twice weekly. To differentiate 3T3-L1 cells into adipocytes, cells were incubated with 100 ng/ml dexamethasone, 100 μg/ml 3-isobutyl-1-methylxanthine, and 1 μg/ml insulin for 4 days, followed by 1 μg/ml insulin for an additional 4–8 days.3T3-L1 cells were obtained from American Type Culture Collection (Manassas, VA) and grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) fetal calf serum (Irvine Scientific, Santa Ana, CA), 1 mM sodium pyruvate, 100 μg/ml streptomycin sulfate, and 100 units/ml penicillin. Cells were cultured at 37°C with 5% COCell lysates were prepared with 20 mM Tris pH 7.4, 150 mM NaCl (TBS) containing 1% (v/v) Triton-X100. For leptin, culture supernatants were concentrated by mixing with 3 volumes ice cold acetone, incubating at -20°C for 1 h, followed by centrifugation at 10,000 × g at 4°C for 15 min. Protein pellets were resuspended with SDS-PAGE sample buffer supplemented with 2% (v/v) β-mercaptoethanol. Proteins were then separated by SDS-PAGE and transferred to Immobilon-P (Millipore, Billerica, MA) using a wet tank transfer system (BioRad, Hercules, CA). Membranes were blocked with TBS, 0.1% (v/v) Tween-20, 5% (w/v) non-fat dry milk for 20 minutes at 23°C and incubated with the indicated antibody for 2 h at 23°C. Membranes were washed three times (10 min each) with TBS, 0.1% (v/v) Tween-20, and bound antibodies were detected with species-specific HRP-conjugated secondary antibodies (1:3000, BioRad) followed by chemiluminescence detection according to the manufacturer's instructions (Pierce, Rockford, IL). Images were captured using a Syngene GeneGnome system equipped with a Peltier-cooled 16-bit CCD camera and saturation detection. Densitometric analysis was performed using Scion Image, version 4.0.2.125I as previously described [125I-RAP-GST (500 ng/ml) diluted into 20 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1% (w/v) bovine serum albumin (buffer A) at 4°C for 3 h in the presence or absence of a 50-fold molar excess of unlabeled RAP-GST. Unbound 125I-RAP-GST was removed by rinsing cells three times with cold buffer A after which cells with bound ligand were solubilized with 0.1 N NaOH. Solubilized proteins were added to EcoLume  and subjected to scintillation counting (Packard Tri-Carb 1600CA, 73% efficiency for 125I). Results were normalized to total cellular protein (BCA Protein Assay, Pierce, Rockford, IL). Specificity was determined as the difference between total binding (without competition) and non-specific binding (non-competable) [125I-labeled ligand. All data points represent averages of duplicates or triplicates with standard errors of <5%.RAP-GST was purified  and labeescribed . Differepetable) . The act2 for 3 h with 500 ng/ml 125I-RAP-GST diluted into DMEM containing 1% (w/v) bovine serum albumin in the presence or absence of a 50-fold molar excess of unlabeled RAP-GST. Media was then removed and processed for trichloroacetic acid (TCA) precipitation [Differentiated 3T3-L1 cells were incubated at 37°C/5% COpitation . TCA-sol2 35SO4 for 20 h at 37°C/5% CO2. Cells were detached by incubating with phosphate buffered saline containing 5 mM EDTA for 20 min at 23°C. Following centrifugation to pellet cells (1000 × g, 4°C, 10 min), they were lysed by incubation with solubilization buffer (100 mM Tris, pH 7.5, 150 mM NaCl, 8 M urea) for 1 h at 4°C. Insoluble material was removed by centrifugation (10,000 × g, 4°C, 5 min) and supernatant was batch adsorbed onto 0.5 ml Macro-Prep DEAE Support (BioRad) (pre-washed with solubilization buffer) with gentle agitation for 1 h at 4°C. DEAE resin was applied to a 1.0 cm × 5.0 cm column and washed with 10 column volumes solubilization buffer followed by 10 column volumes 100 mM Tris, pH 7.5, 150 mM NaCl. Bound proteins were eluted with 100 mM Tris, pH 7.5, 1.0 M NaCl and 120 μl fractions were collected. Fifteen μl from each fraction was mixed with 3 ml EcoLume and subjected to scintillation counting. Peak fractions from DEAE enrichment of proteoglycans obtained from labeled differentiated adipocytes  or chondroitinase ABC (5 units/ml) or both for 20 h at 37°C. The material was applied to a 1.0 cm × 12 cm Sephacryl S-200 HR (Amersham Biosciences, Piscataway, NJ) gel filtration column and eluted with 20 mM Tris, pH 7.4, 150 mM NaCl. 350 μl fractions were collected of which 200 μl was mixed with 3 ml EcoLume and analyzed by scintillation counting.3T3-L1 pre-adipocytes and differentiated adipocytes grown in 100 mm tissue culture dishes were incubated with sulfate-free DME containing 10% (v/v) fetal calf serum and 125 μCi/ml NaNew Zealand White rabbits were placed on a high-fat chow diet (10% peanut oil/1% cholesterol) for a minimum of 4 d, blood was drawn into in 1 mM EDTA and centrifuged at 2000 × g, 15 min to remove cells. Chylomicrons were floated by centrifuging plasma at 100,000 × g for 10 min and removed by pipetting. Plasma was then mixed with OptiPrep™  and centrifuged at 350,000 × g for 3 h (SW55Ti rotor) with slow acceleration and deceleration. VLDL particles (density of 1.006 g/ml) were removed from the top of the gradient by pipetting. Purified VLDL was analyzed by SDS-PAGE and Coomassie R staining to confirm the presence of apoB100 (515 kD) and apoE (35 kD). Animal protocol (#2032) was approved by the University of New Mexico, Health Sciences Center Laboratory Animal Care and Use Committee. For DiI labeling, a working stock of 3 mg/ml was made in dimethylsulfoxide and 0.15 mg was slowly added to 1.67 mg VLDL (in 1.9 ml) with vortexing to rapidly mix. The mixture was then wrapped in foil and incubated for 8 h at 37°C. Unbound DiI was removed from DiI-labeled VLDL by OptiPrep gradient centrifugation as described above.3T3-L1 cells were plated on glass coverslips and incubated with differentiation reagents as described above. After conversion to mature adipocytes, cells were rinsed twice with DMEM and incubated with DiI-VLDL (4 μg/ml) and apolipoprotein E (3 μg/ml) diluted into DMEM in the presence or absence of either heparin (500 μg/ml) or RAP-GST (50 μg/ml). After 3 h at 37°C, cells were rinsed with phosphate buffered saline, fixed with 1.5% (w/v) paraformaldehyde for 30 min, and mounted in Gelvatol (Air Products, Allentown, PA) containing 1 mg/ml p-phenylenediamine. Cells were observed with a Zeiss Axioskop microscope equipped for epifluorescence. Images were capture with a Hamamatsu digital/video camera and AxioVision software.Treated and non-treated 3T3-L1 cells (as indicated in figure legends) were cultured overnight in serum-free DMEM without phenol red. Media was removed and combined with an equal volume of 100 mM sodium phosphate buffer, pH 7.2, 150 mM NaCl, 0.5% (v/v) Triton-X100. p-nitrophenyl butyrate in acetonitrile was added to a final concentration of 0.5 mM and absorbance at 400 nm was recorded every 10 s for 5 min using a Genesys UV Spectrophotometer. A standard curve for LPL activity was generated by plotting absorbance values obtained with varying concentration of purified LPL (from bovine milk). Quantitation of LPL activity in the individual media samples was determined from the standard curve.Cells were fixed with 10% (v/v) formaldehyde in phosphate buffered saline for 1 h at 23°C, rinsed twice with water, then stained for 2 h at 23°C with 0.1% (w/v) Oil Red O in 75% (v/v) isopropanol, followed by rinsing twice with water to remove unincorporated dye. Stained cells were viewed and photographed using a Zeiss AxioVert microscope with phase contrast optics and Hamamatsu digital/video camera. For quantitation, cells were dried for 2 h at 37°C, followed by incubation with 100% isopropanol for 15 m at 23°C to extract bound dye. Solubilized dye was then quantitated by spectrophotometry in a BioRad Model 680 Microplate Reader by measuring absorbance at 510 nm. Statistical significance was determined by performing a paired t-test.ApoE = apolipoprotein EHSPG = heparan sulfate proteoglycanVLDL = very low density lipoproteinVLDL-R = VLDL receptorLRP = low density lipoprotein receptor-related proteinSyn-1 = Syndecan-1FATP1 = fatty acid transport protein-1RAP = receptor associated proteinLPL = lipoprotein lipaseDiI = 1,1'-dictadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate4-MUmb = 4-methylumbelliferyl-β-D-xylopyranosidepNP-Xyl = p-nitrophenyl-β-D-xylopyranosidebFGF = basic fibroblast growth factorLCW carried out the majority of studies and drafted the manuscript. SC performed the xyloside titration study and DN performed the LPL assays. RAO provided the original conceptual framework for the study, carried out pilot experiments, participated in the experimental design and finalized the manuscript for submission. All authors read and approved the final version.


The fusion protein RUNX1-CBFA2T1 associated with t-positive acute myeloid leukaemia is a potent inhibitor of haematopoetic differentiation. The role of RUNX1-CBFA2T1 in leukaemic cell proliferation is less clear. We examined the consequences of siRNA-mediated RUNX1-CBFA2T1 depletion regarding proliferation and clonogenicity of t-positive cell lines.The t-positive cell line Kasumi-1 was electroporated with RUNX1-CBFA2T1 or control siRNAs followed by analysis of proliferation, colony formation, cell cycle distribution, apoptosis and senescence.Electroporation of Kasumi-1 cells with RUNX1-CBFA2T1 siRNAs, but not with control siRNAs, resulted in RUNX1-CBFA2T1 suppression which lasted for at least 5 days. A single electroporation with RUNX1-CBFA2T1 siRNA severely diminished the clonogenicity of Kasumi-1 cells. Prolonged RUNX1-CBFA2T1 depletion inhibited proliferation in suspension culture and G1-S transition during the cell cycle, diminished the number of apoptotic cells, but induced cellular senescence. The addition of haematopoetic growth factors could not rescue RUNX1-CBFA2T1-depleted cells from senescence, and could only partially restore their clonogenicity.RUNX1-CBFA2T1 supports the proliferation and expansion of t-positive leukaemic cells by preventing cellular senescence. These findings suggest a central role of RUNX1-CBFA2T1 in the maintenance of the leukaemia. Therefore, RUNX1-CBFA2T1 is a promising and leukaemia-specific target for molecularly defined therapeutic approaches. The chromosomal translocation t8;21) , which is associated with 10–15% of all cases of acute myeloid leukaemia, fuses the DNA binding domain of the transcription factor RUNX1  to the almost complete open reading frame of CBFA2T1  [  provides a new approach to investigate the functions of oncogenes in the development of cancer, thereby complementing other approaches such as ectopic (over-) expression -36. We a3 . Here, wThe siRNAs siAGF1 and siAM targeting the fusion site of the RUNX1-CBFA2T1 mRNA, the mismatch control siAGF6, and the unrelated controls targeting luciferase (siGL2) (for sequences see ref. 13) or MLL-AF4  were synthesized by either Alnylam Europe AG (Kulmbach, Germany) or MWG Biotech (Ebersberg, Germany) and hybridized as described [6/ml in 4 mm electroporation cuvettes. SiRNAs were added immediately before electroporation. If not otherwise indicated, the siRNA concentration during electroporation was 100 nM. Electroporations were performed using a rectangle pulse EPI 2500 electroporator . Parameters were 330 V and 10 ms (milliseconds) for Kasumi-1 cells, and 350 V and 10 ms (milliseconds) for all other cell lines. Fifteen minutes after electroporation, cells were diluted twenty fold in culture medium and incubated at 37°C, 5% CO2 and 92% humidity. Using this protocol, we routinely observe less than 10% of dead cells after electroporation with an siRNA transfection efficiency close to 100% [The t-positive acute myeloid leukaemia (AML) cell lines Kasumi-1 (Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ) No. ACC 220)  and SKNO to 100% ,42,43.To obtain total cell lysates, cells were washed with phosphate-buffered saline, lysed in Urea lysis buffer (9 M urea, 4% (w/w) 3-[(3-Cholamidopropyl)-dimethylammonio]-propansulfonat (CHAPS), 1% (w/w) Dithiothreitol) and treated for 30 min on ice with 2.5 u/ml Benzonase (Merck, Darmstadt, Germany). Total (10–20 μg) and nuclear lysates (10 μg) were analyzed as described . Band in- (Promega, Heidelberg, Germany) as suggested by the manufacturer. Real time PCRs were performed using Sybr-Green Mix (Applied Biosystems, Warrington, UK), 300 nM primers and 20% v/v diluted RT reaction mix using standard conditions on a 7000 Sequence detection system (Applied Biosystems, Foster City, CA, USA). The primer sequences for STAT1 (5'-CAT CAC ATT CAC ATG GGT GGA (forward primer) and 5'-GGT TCA ACC GCA TGG AAG TC (reverse primer)), for CD34 (5'-TCC AGA AAC GGC CAT TCA G (forward primer) and 5'-CCC ACC TAG CCG AGT CAC AA (reverse primer)), for G-CSF (5'-CCC ACC TTG GAC ACA CTG C (forward primer) and 5'-AGT TCT TCC ATC TGC TGC CAG (reverse primer)) and for GAPDH (5'-GAA GGT GAA GGT CGG AGT C (forward primer) and 5'-GAA GAT GGT GAT GGG ATT TC (reverse primer)) were designed with Primer-Express software (Applied Biosystems, Foster City, CA, USA).Total RNA was isolated using the RNeasy Kit (Qiagen, Hilden, Germany). Reverse transcription was performed with random hexamers using MMLV-RT, RNase HTo determine proliferation rates and doubling times, cells were counted using trypan blue. For single cell expansion assays, 24 h after electroporation cells were diluted to a density of 10 cells/ml and plated in 100 μl aliquots into 96 well tissue culture plates. Colony formation assays were performed 24 h after electroporation with a density of 5,000 cells/ml in semisolid medium . Colonies were counted 14 days after plating. The cytokine concentrations were 20 ng/ml for G-CSF and GM-CSF in the cell cycle and senescence assays, and 10 ng/ml for all growth factors in the colony formation assays.6 cells were suspended in 200 μl citrate buffer (250 mM sucrose, 40 mM sodium citrate pH 7.6) followed by the addition of 800 μl staining solution  NonIdet P40, 500 μM EDTA) and 10 μl boiled RNase A (10 g/l). Cells were kept for 30 minutes on ice and analyzed by flow cytometry . Data analysis was performed with FCSPress 1.3 .Cell cycle analysis was performed as described . Four da for data analysis.For quantification of apoptotic cells, cells were stained with FITC-labeled annexin V as suggested by the supplier (Bender, Vienna, Austria). CD34 expression was monitored by staining with anti-human CD34-FITC (clone 581, BD Pharmingen #555388, Heidelberg, Germany). Analysis was performed by flow cytometry  using FCSPress 1.3 Electroporation of t-positive cells with the RUNX1-CBFA2T1 siRNA siAGF1, but not with the mismatch control siRNA siAGF6, resulted in a three- to fourfold reduction of RUNX1-CBFA2T1 fusion protein in both Kasumi-1 cells fig. . This fiTransfection with siRNAs may induce an interferon response leading to non-specific effects on cellular processes such as proliferation or differentiation . Since SIn comparison to mock-transfected cells, electroporation with RUNX1-CBFA2T1 siRNAs resulted in a ten- to twenty fold decreased clonogenicity in both single cell expansion (data not shown) and colony formation assays -negative myeloid leukaemia cell lines MV4-11, NB4, HL60, U937 and K562 was not affected upon electroporation with RUNX1-CBFA2T1 siRNA (data not shown), which argues against a general, unspecific effect of the siRNAs used in this study on leukaemic clonogenicity. Therefore, the application of RUNX1-CBFA2T1 siRNAs is sufficient to inhibit the clonogenic growth of Kasumi-1 cells, indicating that RUNX1-CBFA2T1 is essential for the clonogenicity of these leukaemic cells.To examine the effects of RUNX1-CBFA2T1 on the progenitor status of t-positive leukaemic cells, we analyzed the CD34 expression levels in siRNA-treated Kasumi-1 cells. When compared to mock-transfected cells, depletion of RUNX1-CBFA2T1 caused a twofold decrease in CD34 surface expression 7 days after electroporation . Two or more consecutive electroporations with RUNX1-CBFA2T1 siRNA caused a reduction of the amount of S phase cells by 50% and a 30% reduction in G2/M phase cells, with a corresponding increase of the G1/G0 fraction by 20% fig. . TreatmeKip1), a general inhibitor of Cyclin-CDK complexes and, thereby, of cell cycle progression. Two electroporations with RUNX1-CBFA2T1 siRNA caused a twofold increase in CDKN1B protein levels in RUNX1-CBFA2T1-depleted cells, but not in control cells (fig. The changes in the cell cycle distribution are associated with changed expression levels of CDKN1B (p27lls fig. . TherefoA possible reason for the observed inhibition of proliferation is the induction of apoptosis upon suppression of RUNX1-CBFA2T1. We addressed this possibility by examining the amount of apoptotic cells by annexin V staining and of hypodiploid (subG1) cells by flow cytometry. Electroporation of Kasumi-1 cells together with the RUNX1-CBFA2T1 siRNA siAGF1 neither increased the amount of hypodiploid cells nor the amount of annexin V positive cells when compared to control cells. Instead, after siAGF1 treatment we observed a slight but reproducible decrease in the amount of apoptotic cells in both assays. This decrease was more pronounced after two consecutive electroporations with siRNAs. Repeating the siAGF1 electroporation after 4 days resulted in a threefold decrease of the amount of annexin V stained cells fig.  indicatiThe observed G1 arrest upon RUNX1-CBFA2T1 depletion could be either reversible or irreversible. An irreversible G1 arrest is a hallmark of cellular senescence. To address the question of the nature of the G1 arrest, and to analyze a possible influence of RUNX1-CBFA2T1 on cellular senescence, we stained siRNA- and mock-treated cells for senescence-associated β-galactosidase activity. After two or more electroporations with RUNX1-CBFA2T1 siRNA, but not with control siRNA, a significant fraction of the Kasumi-1 cells stain positive for β-galactosidase fig. . Cell coA possible mechanism of how RUNX1-CBFA2T1 prevents cell cycle arrest and senescence is the autocrine production of growth factors. Kasumi-1 cells express, for instance, G-CSF fig. , but notDepletion of RUNX1-CBFA2T1 for 16 days reduced the fraction of S phase cells twofold compared to control cells. This decrease is independent of G-CSF or GM-CSF fig. . When anFinally, we examined the effects of several haematopoetic growth factors on colony formation of siRNA-treated Kasumi-1 cells. Supplementing the semisolid medium with the corresponding growth factors increased colony numbers for both control cells and RUNX1-CBFA2T1-depleted cells fig. . G-CSF eTaken together, the addition of G-CSF or GM-CSF delays, but does not prevent the G1 arrest in RUNX1-CBFA2T1-depleted cell. Furthermore, senescence is not inhibited by these growth factors. In line with these findings, neither of these growth factors completely restored the impaired clonogenicity of such cells. These results indicate that neither G-CSF nor GM-CSF can rescue leukaemic cells suffering of siRNA-mediated RUNX1-CBFA2T1 depletion.3 receptor [Arf [ex vivo and in vivo [The leukaemic fusion protein RUNX1-CBFA2T1 inhibits haematopoetic differentiation by repressing gene expression via recruitment of histone deacetylases -7, and breceptor -11. In areceptor ,46, in stor [Arf . The not in vivo ,21. In aWe show that RUNX1-CBFA2T1 also supports proliferation, CD34 expression and colony formation in the t-positive leukaemic cell line Kasumi-1. Thus, RUNX1-CBFA2T1 functions are similar in HSCs and in Kasumi-1. In the latter, support of proliferation is not caused by an inhibition of cell death. Instead, as observed with many other cell types ,46, RUNXKip1 (CDKN1B) during RUNX1-CBFA2T1 depletion are in line with the recently demonstrated central role of this CDK inhibitor in the establishment of senescence [The reduced proliferation of Kasumi-1 cells upon RUNX1-CBFA2T1 depletion is paralleled by a cell cycle arrest in G1. Particularly from a therapeutic point of view, it is important to know, whether this G1 block will be relieved by recovering RUNX1-CBFA2T1 expression, or by growth factors, or whether this arrest is irreversible. We observe that RUNX1-CBFA2T1 depleted cells show enhanced levels of senescence-associated β-galactosidase activity, a hallmark of cellular senescence. Therefore, the inhibition of G1 to S transition is associated by the induction of senescence indicating that this G1 arrest is irreversible. Interestingly, the elevated levels of p27nescence ,50.Arf [Arf and of its human homologue, p14Arf [Furthermore, RUNX1 was shown to induce senescence in murine embryonic fibroblasts, probably by inducing p19Arf . Since R, p14Arf , an incr3 [An important point to consider is a possible relation between the effects of RUNX1-CBFA2T1 on clonogenicity, proliferation and senescence, and its anti-differentiation activity. For instance, increased β-galactosidase activity may also be associated with late monocytic differentiation . Moreove3 . However3 , and datin vivo might interfere with the development and maintenance of leukaemia. However, a remaining possibility might be that the stroma environment with its complex mixture of growth factors and cell-cell interactions may be able to rescue RUNX1-CBFA2T1-depleted cells. We are currently investigating this question both in cell culture and in vivo.The haematopoetic growth factors G-CSF and GM-CSF were shown to support clonogenicity and proliferation in Kasumi-1 cells . HoweverA major point of concern for siRNA applications is the specificity of the corresponding siRNA . ReasonsIn summary, we show that RUNX1-CBFA2T1 does not only inhibit the myeloid differentiation , but is HPV is an employee of Alnylam Europe AG, which also provided some of the siRNAs used in this study.NM performed the proliferation, senescence and cell cycle assays. BD analyzed the clonogenicity of growth-factor treated Kasumi-1 cells and of t negative cell lines. HR performed the immunoblot analyses and was involved in the analysis of clonogenicity and apoptosis. CC examined the expression levels of CD34 and of STAT1. HPV, AG, GH and AN participated in the coordination of the study. JK and OH conceived and designed the experiments. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


By aligning probe sequences with expressed sequence tags (ESTs) in NHP, inter-species conserved (ISC) probesets, which have two or more probes complementary to ESTs in NHP, were identified on human GeneChip® platforms. The utility of human GeneChips® for the assessment of NHP expression patterns can be effectively evaluated by analyzing the hybridization behaviour of ISC probesets. Appropriate normalization methods were identified that further improve the reliability of human GeneChips® for interspecies (human vs NHP) comparisons.While researchers have utilized versions of the Affymetrix human GeneChip® platforms (U133Plus2.0, U133A, U133B, U95Av2, U95B, Focus and HuGeneFL) were identified for both monkey and chimpanzee. Expression data was generated from peripheral blood mononuclear cells (PBMCs) of 12 human and 8 monkey (Indian origin Rhesus macaque) samples using the Focus GeneChip®. Analysis of both qualitative detection calls and quantitative signal intensities showed that intra-species reproducibility (human vs. human or monkey vs. monkey) was much higher than interspecies reproducibility (human vs. monkey). ISC probesets exhibited higher interspecies reproducibility than the overall expressed probesets. Importantly, appropriate normalization methods could be leveraged to greatly improve interspecies correlations. The correlation coefficients between human (average of 12 samples) and monkey (average of 8 Rhesus macaque samples) are 0.725, 0.821 and 0.893 for MAS5.0 (Microarray Suite version 5.0), dChip and RMA (Robust Multi-chip Average) normalization method, respectively.ISC probesets in each of the seven Affymetrix GeneChip® platforms to assess the expression profiles of NHP for intra-species studies. Caution must be taken for interspecies studies since unsuitable probesets will result in spurious differentially regulated genes between human and NHP. RMA normalization method and ISC probesets are recommended for interspecies studies.It is feasible to use Affymetrix human GeneChip An underlying assumption in such studies is that transcripts of humans and NHP are highly conserved, and probe sequences designed to detect human genes will detect their orthologs in NHP samples. It is estimated that chimpanzees (Pan troglodytes) and humans shared 98.77 % DNA similarity [® for NHP expression analysis.Microarray studies on non human primates (NHP) have been used to address viral pathogenesis ,2, neuromilarity  comparedmilarity ,12, and ® platforms for NHP expression profiling have robustly addressed the quantitative aspects of cross platform performance. Vahey and colleagues [® and demonstrated that there was no significant difference in the dynamic range of the raw fluorescence distribution for equivalent amounts of human cRNA and macaque cRNA hybridized to the chip. Chismar and colleagues [® platform and compared the expression patterns of humans with that of the rhesus macaque. They concluded that the percentage of 'present' calls observed in the transcriptome of macaque brain is lower than that of human brain, and that this is especially true for genes with lower signal intensity. Caceres and colleagues [® platforms for the study of expression profiles in NHP is to employ a sequence analysis approach.Few published studies employing human GeneChiplleagues  used thelleagues  used thelleagues  used the® platforms to assess expression patterns in NHP samples by: a) identifying ISC probesets based on sequence analysis; b) assessing intra (within NHP species)- and interspecies (between NHP and human samples) reproducibility of GeneChip® data; and c) applying appropriate normalization methods to improve interspecies reproducibility.In this study, we address the power of human GeneChip® hybridizes with the transcriptome of a NHP, there are three possible outcomes: 1) it hybridizes with the ortholog of the NHP; 2) it cross-hybridizes with a non-ortholog transcript, or 3) it fails to hybridize due to sequence divergence. In Affymetrix GeneChip® system, a probeset is composed of 11–20 probes and each probe is a 25-mer oligo. We identified probes on the human GeneChip that are complementary to ESTs in NHP postulating that these probes would hybridize most optimally with the transcripts of NHP. We defined a probeset as an ISC probeset if it had at least two complementary probes. The rationale used to define the criterion that defines an ISC probeset is described in the methods section. The procedure used to generate ISC probesets is shown in Figure ® platforms (U133Plus2.0, U133A, U133B, U95Av2, U95B, Focus and HuGeneFL) were generated for both monkey and chimpanzee. Detailed information about each ISC probeset such as probe sequence, GenBank accession and the position and degree of matching is provided in the supplemental materials (Macaca mulatta) than for chimpanzee (Pan troglodytes). This is not because monkey EST sequences are more similar to human sequences than chimpanzee EST sequences, but because we have a much greater amount of EST sequences available for monkey. At the time of writing this manuscript, there were 33,474 monkey ESTs available, while there were only 6,943 ESTs available for chimpanzee. As the number of defined ESTs will increase in the future, additional ISC probesets could be identified for both monkey and chimpanzee using this method.When a probe sequence on the human GeneChipaterials . Table 1®s, there are three probesets (217028_at, 211919_s_at and 209201_x_at) that target the gene CXCR4 at different positions in its transcript. In order to address this redundancy issue, we converted the number of probesets into the number of unique UniGene clusters based on the GeneChip® annotation file provided by Affymetrix Website [® and the U133Plus2.0 GeneChip® have the lowest and highest frequency of redundant probesets for a given gene, respectively.It is not uncommon, especially in the U133Plus2.0 platform, that multiple probesets target the same gene. For example, in the U133A and the U133 Plus 2.0 GeneChip Website . While a® from Affymetrix and covers the human genome most extensively. Figure The U133Plus2.0 is the most current version of human GeneChip® data observed in intra- and interspecies samples. The intra-species reproducibility is displayed in Figure ® for NHP intra-species studies.The qualitative detection call (present / absent) output from MAS5.0 was the initial approach used to examine the reproducibility of GeneChip® platforms are used to compare the expression pattern of humans with those of NHPs, care must be taken in the interpretation of data. If we consider a probeset as being expressed when 50% or more of replicates have present calls, then 3445 (2059+1386) and 2321 (2059+262) probesets are expressed in the PBMC fraction of humans and monkeys, respectively  was created. Probesets that are not expressed in human PBMCs were excluded in this analysis. Pair-wise correlation coefficients were calculated for all 20 samples . The correlation coefficients were visualized using heat spectrum graphs where colors ranging from red to white correspond to correlation coefficients of 0.5 to 1.0, respectively. In figure To assess the intra- and interspecies reproducibility of GeneChip® data variation [® data at the probe level using a non-linear algorithm while MAS5.0 normalizes data at probeset level using linear scaling. Sequence divergence usually leads to one or very few probes in a probeset being problematic while the majority of probes in that probeset may still work reasonably well. If the variation generated from these problematic probes were normalized, the interspecies reproducibility should improve. Figure Different normalization methods have been shown to significantly affect GeneChipariation -17. We cariation -16 and d® expression data as applied to the derivation of meaningful expression profile data from NHP. The utility of the human Affymetrix GeneChip® for the assessment of expression profiles in NHP depends on the experimental design and on the approach to data normalization and analysis. Our observations suggest that: 1) it is feasible to use the human GeneChip® in the evaluation of expression profiles of NHP samples for intra-species comparisons; 2) use of ISC probesets and RMA normalization are recommended for interspecies studies; and 3) with the increasing amount of ESTs of NHP, additional ISC probesets (and perfect probesets) will be identified in the near future.This paper presents a comprehensive analysis of probe sequences and GeneChipMacaca mulatta) and chimpanzee (Pan troglodytes) were downloaded from NCBI website [Affymetrix GeneChip probe sequences were downloaded from Affymetrix website . The EST website .® probe sequences and monkey /chimpanzee EST sequences. The length of a probe sequence is always 25 nucleotides while the number of probes in a probeset varies from 11 to 20 depending on GeneChip® platforms  in comparison with an RT-PCR experiment, the primer length is equivalent to our probe length, and two primers (one forward and one backward) usually generate a unique sequence in a whole genome; 3) a probe sequence on the Affymetrix GeneChip® is a well designed sequence with a single probe hybridizing with a unique transcript in whole transcriptome; and 4) since the EST sequences in NHP are very limited so far, most of them do not cover whole transcript such that a false negative could be generated if we require all the probes in a probeset being complementary to known ESTs. In order to convert probeset IDs to UniGene IDs and map them onto chromosomes, probeset annotation files were downloaded from Affymetrix website [Stand alone BALST program was downloaded from NCBI website . Perl sc website . No animRhesus macaque) was collected and peripheral blood mononuclear cells (PBMCs) were separated by Histopaque-Ficoll (Sigma) gradient centrifugation. RNA preparation, Hybridization, staining and scanning of the GeneChip® was carried out as described by Vahey et al. [®. Signal values and detection calls (present or absent) for all samples were determined by using MAS5.0 . Signal values were scaled to the default target signal intensity of 500). A matrix of detection calls  and a matrix of signal intensities for all samples across all probesets were constructed. A gene must exhibit 50% or more of 'present' calls in all samples to be considered 'expressed'. In this study, an expressed probeset in human is a probeset that has 6 or more present calls among 12 human samples. Similarly, an expressed probeset in monkey means there were 4 or more present calls among 8 monkey samples. The signal intensities output from MAS5.0 were log2 transformed. Model-based normalization was performed using dChip version 1.3 [rma function in the package was used at its default setting, that is, 'RMA' background correction, 'quantile normalization', 'PM only model' and 'median polish summarization'. By default, the signal intensities were already log2 transformed.Briefly, peripheral blood from healthy human and NHP  and set color scales between 0.5 (red) and 1.0 (white).Intra- and interspecies correlation coefficients of signal intensities were calculated by built in function 'n 1.9.0. . VisualiNHP: non human primateEST: expressed sequence tagISC: inter-species conservedRMA: robust multi-chip averageMAS5.0: Microarray suite version 5.0ZW developed the original hypotheses, performed the bioinformatics analyses to test them and drafted the manuscript. MV provided critical input on design and execution of the laboratory experiments and with ZW interpreted the data sets and revised the manuscript. ML conducted all aspects of the animal handling including the harvest of well characterized primary samples. MN and AA are technical staff who extracted the nucleic acid and performed the laboratory portions of the microarray experiments. All authors read and approved the final manuscript.Macaca mulatta) in GeneChip® platforms Plus2.0, U133A and U133B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are three worksheets in this file. Worksheet 1, 2 and 3 are ISC probesets for monkey (Click here for fileMacaca mulatta) in GeneChip® platforms Focus, FL, U95Av2 and U95B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are four worksheets in this file. Worksheet 1, 2, 3 and 4 are ISC probesets for monkey (Click here for filePan troglodytes) in GeneChip® platforms Plus2.0, U133A and U133B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are three worksheets in this file. Worksheet 1, 2 and 3 are ISC probesets for chimpanzee (Click here for filePan troglodytes) in GeneChip® platforms Focus, FL, U95Av2 and U95B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are four worksheets in this file. Worksheet 1, 2, 3 and 4 are ISC probesets for chimpanzee (Click here for file


Patients suffering from diseased and injured organs may be treated with transplanted organs. However, there is a severe shortage of donor organs which is worsening yearly due to the aging population. Scientists in the field of tissue engineering apply the principles of cell transplantation, materials science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Both therapeutic cloning (nucleus from a donor cell is transferred into an enucleated oocyte), and parthenogenesis (oocyte is activated and stimulated to divide), permit extraction of pluripotent embryonic stem cells, and offer a potentially limitless source of cells for tissue engineering applications. The stem cell field is also advancing rapidly, opening new options for therapy. The present article reviews recent progress in tissue engineering and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure. Cells may be heterologous (different species), allogeneic , or autologous . Autologous cells are preferred because they will not evoke an immunologic response and thus the deleterious side effects of immunosuppressive agents can be avoided.The goal of tissue engineering is to repair organ pathologies such as those acquired congenitally or by cancer, trauma, infection, or inflammation. It is based upon the foundations of cell transplantation and materials science. Tissue can be engineered 1) 2 to one that covers a surface area of >4000 m2  within 8–14 weeks. These studies indicate the possibility of collecting autologous bladder cells from human patients, expanding them in culture, and returning them to the human donor in sufficient quantities for reconstructive purposes [The ideal autologous cells can often be found within the organ itself. These cells (committed precursors) may be isolated, expanded and transplanted back into the same patient, thus representing an autologous transplantation resource. Previously, urothelial cells could be grown in the laboratory setting with only limited expansion. Several protocols were developed over the last 20 years which identified the undifferentiated cells and kept them undifferentiated during their growth phase -4. Usingpurposes -11. Majoi.e. pancreas, liver). Stem cells are envisioned as being an alternate source of cells from which the desired tissue can be derived. Human embryonic stem cells (HESC) can be derived from discarded non transferred embryos and have the advantage of being pluripotential  and able to self-renew indefinitely. However, their clinical application is limited because they represent an allogeneic resource and thus their use would require high dose immunosuppressant therapy.While autologous cells are recognized as the ideal transplantation resource, many patients with end-stage organ disease are unable to yield sufficient cells for expansion and transplantation. Furthermore, some primary autologous human cells cannot be expanded from particular organs (New stem cell technologies such as somatic cell nuclear transfer (therapeutic cloning) and parthenogenesis offer an exciting alternative to create an inexhaustible supply of ESC that can differentiate into all cell types of the embryo, while not being rejected by the patient's immune system. Although many tissues have been created with ESC, they are not used clinically because of an inability to control differentiation. Hence, their ability to form multiple tissue types also becomes their limitation. New genomics and bioinformatics technologies have and will continue to offer new insights into the understanding of ESC growth and differentiation and their application to engineering tissues. In the near future, these new technologies will allow for the generation of an unlimited supply of any cell type in the body.ex vivo, ESC can potentially be the ideal resource for tissue engineering because of two fundamental properties, 1) the ability to self-renew indefinitely, and 2) the ability to differentiate into all three germ layers.The political and ethical controversy surrounding stem cells began in 1998 with the creation of HESC derived from discarded, non-transferred human embryos. The HESn = 7,334) are expected to survive the freeze/thaw process. From this, 25% are expected to develop to blastocyst stage (n = 1, 834). If one assumes a 15% efficiency rate for establishment of a HESC line from blastocysts (as suggested by previous studies [With the current restrictions surrounding HESC work, many proponents of stem cell research have sought to modify the ban to incorporate the thousands of non-transferred frozen embryos resulting from IVF to be used for the creation of more HESC lines. A SART-RAND study identified approximately 400,000 frozen embryos in storage since the late 1970s . However studies ,15), it Somatic cell nuclear transfer (SCNT) entails the removal of an oocyte nucleus followed by its replacement with a nucleus derived from a somatic cell obtained from that patient. Activation with chemicals or electric shock stimulates cell division up to the blastocyst stage at which time the inner cell mass is isolated and cultured, resulting in ESC. This approach is distinct from reproductive cloning because the blasotcyst is not transplanted back to the uterus. Hence, development does not proceed beyond the 100 cell stage. This process also differs from fertilization since no sperm is used in this process. The resulting ESC are perfectly matched to the patients immune system and no immunosuppressants would therefore be required to prevent rejection.adult somatic cell using nuclear transfer [While interest in the field of nuclear cloning remains high since the birth of Dolly 1997), the first successful nuclear transfer was actually reported over fifty years ago by Briggs and King . Cloned 97, the ftransfer , goats [transfer ,22, micetransfer , and pigtransfer -27.A better understanding of the differences between reproductive cloning and therapeutic cloning may help alleviate some of the controversy surrounding these technologies ,29. BannWhile promising, somatic cell nuclear transfer technology has certain limitations requiring further improvement before it can be applied widely in clinical practice. Currently, the efficiency of the overall cloning process is quite low as the majority of embryos derived from animal cloning do not survive after implantation -35. In pIt must be noted that abnormalities have been found in liveborn clones including macrosomia with an enlarged placenta ("large-offspring syndrome") , respiraBos taurus) [in vivo growth. After 12 weeks cloned renal cells were harvested, expanded in vitro, then seeded onto biodegradable scaffolds. The constructs (consisting of cells + scaffolds) were then implanted into the subcutaneous space of the same steer from which the cells were cloned to allow for tissue growth.We applied principles of both tissue engineering and therapeutic cloning in an effort to produce genetically identical renal tissue in an animal model ( taurus) . Bovine ex vivo renal replacement devices are known to be life-sustaining, there are obvious benefits for patients with end-stage kidney disease if such devices could be implanted long-term without the need for an extracorporeal perfusion circuit or immunosuppressive drugs.The kidney is a complex organ with multiple cell types and a complex functional anatomy rendering it one of the most difficult organs to reconstruct ,47. PrevCloned renal cells were seeded on scaffolds consisting of three collagen-coated cylindrical polycarbonate membranes figure . The endChemical analysis of the urine-like fluid (for urea nitrogen/creatinine levels, electrolyte levels, specific gravity, and glucose concentration) revealed that the implanted renal cells possessed filtration, reabsorption, and secretory capabilities. Histological examination of the retrieved implants revealed extensive vascularization and self-organization of the cells into glomeruli- and tubule-like structures. A clear continuity between glomeruli, tubules, and the polycarbonate membrane was noted that allowed the passage of urine into the collecting reservoir figure . ImmunohAs previous studies have confirmed bovine clones harbor mitochondrial DNA (mtDNA) of strictly oocyte origin -57, the in vivo and cytotoxic T lymphocytes expansion in vitro [in vivo and Elispot analysis of interferon-gamma secreting T-cells in vitro. Both analyses revealed that the cloned renal cells showed no evidence of T-cell response, suggesting that rejection will not necessarily occur in the presence of oocyte-derived mtDNA  is production of offspring by a female with no genetic contribution from a male and without meiotic chromosome reduction. The process is common reproductive strategy among insects such as aphids, flies, ants, and honeybees, but is also known to occur in vertebrates including lizards, snakes, fish, birds, and amphibians. The first demonstration of artificially-stimulated parthenogenesis in vitro was made by Jacques Loeb (1899), who was able to activate oocytes from sea urchins and frogs by pricking them with a needle or by changing the ambient salt concentration. Pincus (1939) demonstrated parthenogenetic activation of mammalian eggs using temperature and chemical stimuli. Thus far, parthenogenetic activation of eggs has been studied in a variety of mammals including mice, goats, cows, monkeys, and humans. Plachot et al. described parthenogenesis in humans by examining 800 human oocytes and showed that 12 activated parthenogenetically and four underwent normal cleavage[Parthenogenesis  to elevate Ca2+ levels transiently, followed by application of an inhibitor of protein synthesis (cycloheximide) or protein phosphorylation (6-dimethylaminopurine). Success rates and viability appear to be organism dependent. Mouse parthenotes are capable of developing beyond the post-implantation stage in vivo [Callithrix jacchus) have only been shown to implant [2n polar body under the zona pellucida and a 2n protonucleus in the cytoplasm. After chemical activation to mimic the effects of sperm penetration on changes in cellular Ca2+ gradient, the cell fails to complete meiosis II. Instead, the second polar body is never extruded, resulting in a diploid protonucleus derived from two sets of sister chromatids. These chromatids then begin to undergo mitosis resulting in a parthenote manifesting uniparental disomy. Although the derivation of embryonic-like stem cells from oocytes (parthenogenetic stem cells, PSC) is relatively inefficient (perhaps due to complexities of genomic imprinting), when they are differentiated into adult tissues, they appear fully functional.There is no confirmed example of  in vivo ,67; porc in vivo ; primate implant . The rea implant ,71. Inde implant . In mostin vitro in an undifferentiated state for up to 14 months. In vitro, they have been differentiated into cardiomyocyte-like cells, smooth muscle, beating ciliated epithelia, adipocytes, several types of epithelial cells, as well as dopaminergic and serotoninergic neurons. Almost all of these neurons express TUJ1 (beta-tubulin III), and up to 25% of the TUJ1+ cells co-express tyrosine-hydroxylase. This latter enzyme marker is considered diagnostic for catecholaminergic neurons . Furthein vivo, since injection of monkey PSC into immunocompromised mice induces formation of benign teratomas containing tissue derivatives from all three germ layers (ectoderm, endoderm and mesoderm) including cartilage, muscle, bone, neurons, skin, hair follicles, and intestinal epithelia [These observations are recapitulated pithelia ,75. Of ppithelia . The reain vivo tumorigenic event) the de facto acceptance of experiments using teratoma tumor tissue lends some legitimacy to experimentation on parthenotes. These contradictions await reconciliation in a comprehensive ethical framework.To be sure, parthenotes are not free from ethical controversy and are viewed by some in society as artificial entities that in some sense represent 'tampering with nature.' Since a parthenote is analogous to a mature ovarian teratoma , and NP that were further differentiated for 8 days (PSC-neurons). We have identified numerous targets such as receptors and ligands present at each of these distinct time points, and are modifying our culture system in order to improve the quality and quantity of differentiation. Furthermore, we are comparing the gene expression profiles of PSC derived neurons to gene expression profiles of reference neurons. Not only will this provide new insight into the type of neurons that may be generated, but it offers clues into what our stem cell derived neurons might be lacking. We can then go back to the culture system and try to target these specific genes/signaling pathways.Further study of stem cell genomics will give additional insight into pluripotentiality. An understanding of pluripotentiality might allow for a somatic cell to be de-differentiated into an intermediate stage, which could then be expanded, differentiated and transplanted back into the patient. We are presently characterizing the genetic signature of pluripoteniality by analyzing gene expression among primate stem cells derived from a variety of methods (IVF, parthenogenesis, and adult stem cells). By identifying "stemness" genes by comparing undifferentiated stem cells to their differentiated counterpart, and comparing this to stem cells of different origins, a core set of pluripotential target genes may be mapped. Of particular interest are the 1,075 genes that are similarly down-regulated in IVF derived human ESC and monkey PSC. Furthermore, we have detected paternally imprinted genes in our HESC but not in our PSC data sets. From this we conclude that paternal imprinting might not be necessary for pluriopotentiality.Our systems biology approach incorporates the fields of genomics, cell biology, nuclear transfer, and materials science, and utilizes personnel who have mastered the techniques of bioinformatics, cell harvest, culture, expansion, transplantation, as well as polymer design essential for the successful application of these technologies. Experimental efforts are currently underway involving virtually every type of tissue and organ of the human body. Various tissues are at different stages of development with some already being used clinically, a few in pre-clinical trials, and some in the discovery stage. Recent progress suggests that engineered tissues may have an expanded clinical applicability in the future and may represent a viable therapeutic option for those who require tissue replacement or repair.


A review of "On Intelligence", a book exploring brain function by entrepreneur Jeff Hawkins and science writer Sandra Blakeslee Is Michael Moore liberal America's Rush Limbaugh? If so, is he filling a much needed, or a much lamented, gap in turning issues that are really cast in pastel shades into Day-Glo relief? In this hale monograph, Jeff Hawkins (rendered by Sandra Blakeslee) plays exactly this role for theoretical neuroscience. As a pastel practitioner myself, but furtively sharing many of Hawkins' prejudices and hunches about computational modelling in neuroscience, I am caught between commendation and consternation.Hawkins is an engineer, entrepreneur, and scientist who founded and led the companies Palm and then Handspring. He created, against what must have been considerable obstacles, the first widely successful PDA, and continued the development of this platform. He has thus amply earned a bully pulpit. The autobiographical segments of this book detail that, throughout his career, he has been interested in understanding how the brain works, using his substantial knowledge and intuition about the architecture and design of conventional computers as a counterpoint.More recently, Hawkins has generously put his money where his ideas about mentation dictate, founding the Redwood Neuroscience Institute and also funding various conferences and workshops. The institute is dedicated to ‘studying and promoting biologically accurate mathematical models of memory and cognition.’Despite its youth, the Institute already has attracted notable attention as a centre for theoretical neuroscience. Hawkins' quest, and-depending on which statements of the book you read-its endpoint (‘… a comprehensive theory of how the brain works … describ[ing] what intelligence is and how your brain creates it’) or just its tipping point , are the subject here.There are really three books jostling inside the covers. One is the (highly abbreviated) autobiography. The history of modern computing is very brief and  very glorious, and this story is most entertaining. Don't miss the wonderfully faux naive letter from Hawkins to Gordon Moore asking, in 1980, to set up a research group within Intel devoted to the brain. That Hawkins prospered in clear opposition to accepted wisdom is perhaps one of the key subtexts of the book.The second, and rather less satisfying, book is about the philosophy of mind and the history of artificial intelligence and neural network approaches to understanding the brain and replicating cognition. With respect to the fields of artificial intelligence and neural nets, the text seems rather to be fighting yesterday's battles. The importance of learning, flexibility in representation and inference, and even decentralisation of control has been more than amply recognised in the inexorable rise of probabilistic approaches in both fields.With respect to the philosophy of mind, there seems to be something of an enthusiast's disdain for the niceties of philosophical pettifogging, even arguing by assertion. The discussions at the end on creativity and consciousness all seem a bit gossamer. The book is somewhat careless about functionalism, a key doctrine for computational theorists about how brains give rise to minds. According to this doctrine, at least roughly, it is the functional roles of, and functional interactions among, the physical elements of brain that matter, and not their precise physical nature. If you can capture those functional aspects correctly, for instance, in a computer program, then you can (re-)create what's important about mental states. Functionalism licenses a form of inquiry into the computational jobs played by structures in the brain. However, although formally agreeing that ‘there's nothing inherently special or magical about the brain that allows it to be intelligent,’the book slips into statements such as ‘brains and computers do fundamentally different things,’which are, at best, unfortunate shorthand.The book is a little apt to sneak plausible, but misleading, claims under the radar. Just to give one instance, it compellingly compares a six year old hopping from rock to rock in a streambed with a lumbering robot failing to do the same task. However, this is a bit unfair. One of Hawkins' self-denying ordinances is to consider the cortex pretty much by itself. As aficionados of the cerebellum  would be quick to point out, the singular role for the cortex in such graceful behaviour is rather questionable.The third book is what I think is intended to be the real contribution. This contains a (not wholly convincing) attempt to conceptualise the definition of intelligence in terms of prediction rather than behaviour, and then to describe its possible instantiation in the anatomy (and mostly only the anatomy) of the cortex.To situate Hawkins' suggestions, it is instructive to consider current models of how the cerebral cortex represents, and learns to represent, information about the world without being explicitly taught. Being a popular account, the book fairly breezes by these so-called unsupervised learning models see , in whicFirst, where does the structure in the inputs come from? For the sake of concreteness, think of the input as being something like movies on a television screen. Movies don't look like white noise, or ‘snow’, because of their statistical structure. For instance, in movies, pixel activities tend to change rather slowly over time, and pixels that are close to each other on the screen tend to have relatively similar activities at any given time. Neither of these is true of white noise. More technically, movies constitute only a tiny fraction of the space of all possible activations of all the pixels on your screen. They  have a particular statistical structure that the cortex is supposed to extract.What is the cortex supposed to do with this structure? The idea is that the cortex learns to model, or ‘parameterize’, it. Then, the activities of cortical cells over time for a particular input, for example, a particular face in a movie, indicate the values of the parameters associated with that face. Thereby the cortical activities represent the input. The parameters for a face might include one set for its physical structure (e.g., the separation between the eyes and whether it is more round or more square), another set for the expression, and yet others, too.Cortical representations are thus intended to reflect directly the statistical structure in the input. Importantly, for inputs such as movies, this structure is thought to be hierarchical and, concomitantly, to provide an account of the observed hierarchical structure of sensory cortical areas. One source of hierarchical structure in movies is the simple fact that objects (such as the faces) have parts (such as eyes and cheeks) whose form and changes in form over time are interdependent. Another source of hierarchical structure is that the same face can appear in many different poses, under many different forms of illumination, and so on. Pattern theory , one of How does the cortex do all this? Of course, some fraction of this structure was built in over evolution. However, the unsupervised learning tradition concentrates on ontogenic adaptation, based on multiple presented input movies. An additional facet of the lack of supervision is that this adaptation is taken as not depending on any particular behavioural task.Finally, what does this process allow the cortex to do? The whole representational structure is intended to support inference. Crudely, this involves turning partial or noisy inputs into the completed, cleaned-up patterns they imply, using connections between areas in the cortical hierarchy. Construed this way, probabilistic inference actually instantiates a very general form of computation. Crucially, over the course of the development of unsupervised learning methods, it has been realised that the best way to approach the extraction of input structure, and inference with it, is through the language and tools of probability theory and statistics. The same realisation has driven substantial developments in artificial intelligence, machine learning, computer vision, and a host of other disciplines.We can now return to the book. Hawkins compactly sums up his thesis in the following way. ‘To make predictions of future events, your neocortex has to store sequences of patterns. To recall appropriate memories, it has to retrieve patterns by their similarity to past patterns . And finally, memories have to be stored in an invariant form so that the knowledge of past events can be applied to new situations that are similar but not identical to the past.’In fact, to take the latter points first, the sort of auto-associative storage and recall to which Hawkins refers is a theoretically and practically hobbled version of unsupervised learning's probabilistic inference. Invariance is closely related to the deformations we described above in the context of pattern theory.Unsupervised learning has certainly paid substantial attention to sequences of inputs and prediction, and to some good effect. For instance,  speech recognition programs are based on a probabilistic device called a hidden Markov model, which is a key element in a wealth of unsupervised learning approaches to prediction. However, despite heroic efforts, these modelling methods are incapable of capturing the sort of complex structure seen in inputs such as natural languages. They fail on phenomena like long-distance dependencies, for example, the agreement between the cases of subjects and verbs, which are rife. This does tend to offer a vaccine against Hawkins' otherwise infectious optimism.Once place in which Hawkins goes beyond existing unsupervised learning models is in an extension to actions and control, and in an ascription of parts of the model to cortical anatomy. The hierarchical conception of cortex here goes all the way down to primary motor cortex . This allows auto-associative recall of sequences of past inputs and outputs to be used to specify actions that have formerly been successful. The discussion of this possibility is, unfortunately, rather brief. Central issues are omitted, such as the way that planning over multiple actions might happen. Also, the way that value is assigned to outcomes to determine success or failure is not discussed. The latter is widely believed to involve the neuromodulatory systems that lie below the cortex and that the book's cortical chauvinism leads it cheerfully to ignore.By contrast, the book has a rather detailed description of how the model should map onto the anatomy of the cerebral cortex. Like many unsupervised learning modellers, Hawkins is a self-confessed ‘lumper’. He ignores huge swathes of complexity and specificity in cortical structure and connections in favour of a scheme of crystalline regularity. Though this will doubtless irk many readers  constitute a terrible waste of cortical representational power, or, worse, (b) interfere with, or warp, the parameterization of the aspects of the input that are important, making it harder to extract critical distinctions. The book does not address this issue, relying on there being enough predictive power to capture any and all predictions, including predictive characterisation of motor control.Second, although our subjective sense is that we build a sophisticated predictive model of the entire sensory input, experiments into such phenomena as change blindness  show thiAs a final example of a spur to insomnia, unsupervised learners worry that To sum up, in terms of the adage that genius is 1% inspiration and 99% perspiration, the book's enthymematic nature suggests that not quite enough sweat has been broken. Were it 1% inspiration and 99% aspiration, though, then the appealing call to arms for a new generation of modellers should more than suffice.


The Depression Network Study (DeNt) is a multicentre study designed to identify genes and/or loci linked to and/or associated with susceptibility to unipolar depression in Caucasian families. This study presents the method and socio-demographic details of the first 470 affected sibling pairs recruited from 8 different sites in Europe and the United States of America.th edition (DSM-IV) or the International Classification of Diseases 10th edition (ICD-10) criteria for recurrent unipolar depression of moderate or severe degree and who had at least one similarly affected sibling were eligible for the study. Detailed clinical and psychological assessments were undertaken on all subjects including an interview using the Schedules for Clinical Assessment in Neuropsychiatry. Blood samples were collected from all participants to extract DNA for linkage analysis.Probands fulfilling either the Diagnostic and Statistical Manual 4The different sites used different recruitment strategies depending on local health care organisation but despite this there was remarkable similarity across sites for the subjects recruited. Although the Bonn site had significantly older subjects both for age of onset and age at interview, for the sample as a whole, subjects were interviewed in their mid-40s and had experienced the onset of their recurrent depression in their 20s. Preliminary genome screening was able to include 929 out of the 944 subjects (98.4%) typed at 932 autosomal and 544 X chromosome markersThis paper describes the methodology and the characteristics of the subjects from the 414 families included in the first wave of genotyping from the multi-site DeNT study. Ultimately the study aims to collect affected sibling pairs from approximately 1200 families. Genetic risk factors are well established for major affective disorders and a recent twin study has suggested that unipolar depression has a stronger genetic influence than was previously thought. McGuffin and colleagues  have estThe majority of studies suggest a relative risk to siblings (λs) of affective disorder is in the region of 3 .The inheritance of unipolar depression is complex and involves an inter-play of genetic and environmental factors. For unipolar depression these include certain types of severe and threatening life events such as events associated with humiliation or loss ,6.Despite an excess of females to males of about 2 to 1 for unipolar depression, the heritability in a clinically ascertained sample was the same in men and women  while otMost previous linkage studies have been carried out in families identified by a bipolar proband and where unipolar and bipolar relatives are frequently grouped together into a broad definition of affective disorders. Most such studies have focussed on multiple affected extended pedigrees on the assumption that there may be a sub-set segregating a gene of major effect. This approach has been successful in complex disorders such as early onset Alzheimer's disease and breast cancer. However, consistent evidence of major gene effects in bipolar disorder has not been forth-coming. In addiSibling pairs affected with recurrent unipolar depression were recruited from 8 clinical sites: Aarhus, Denmark; Bonn, Germany; Dublin, Ireland; Lausanne, Switzerland; St Louis, USA and London, Cardiff and Birmingham, UK. In addition, where available, parents of the affected sibling pairs were also included in the study.th edition operational criteria (DSMIV) [th edition operational criteria (ICD10), for unipolar depression [Subjects were identified from psychiatric clinics, hospitals, general medical practices and from volunteers responding to media advertisements. Caucasian subjects over the age of 18 were included if they had experienced 2 or more episodes of unipolar depression of at least moderate severity separated by at least 2 months of remission as defined by the Diagnostic and Statistical Manual 4 (DSMIV)  or the Ipression . ProbandSubjects were also excluded if they experienced psychotic symptoms that were mood incongruent or present when there was no evidence of a mood disturbance. Other exclusion criteria were intravenous drug use with a lifetime diagnosis of dependency; depression occurring solely in relation to alcohol or substance abuse or depression only secondary to medical illness or medication, and a clear diagnosis of bipolar disorder, schizophrenia, schizo-affective disorder or acute or transient psychotic disorders in first or second-degree relatives.All subjects were interviewed using the Schedules for Clinical Assessment in Neuropsychiatry (SCAN) ,16. ItemAll interviewers from each site attended a 4-day SCAN training course in the UK. Each site also undertook further inter-rater reliability meetings regularly and annually all interviewers from all sites took part in a joint inter-rater reliability exercise.All sites obtained ethical approval for the DENT study within their own countries and institutions. All study participants gave written informed consent for participation in the study.In addition to the SCAN interview all study participants completed the Eysenck Personality Questionnaire  and a deAt the time of the SCAN interview interviewers obtained 25 ml of whole blood that was collected in 37.5 ml (EDTA containing) monovettes. In addition drops of blood were placed on a Guthrie blood spot card. The blood samples were labelled with a bar code, gently mixed and stored frozen upright in a -20 degree centigrade freezer pending DNA extraction.All phenotypic information from interviews and questionnaires was coded by assigning a number to each subject, and removing any personal identifying information. The same codes were used on the blood sample tubes using a bar code system. The phenotypic information was first entered on an EXCEL spread sheet after which a data file was created using Statistical Procedures for the Social Sciences (SPSS) version 10 for Windows for the statistical analyses.All the interviewers from all sites took part in a joint inter-rater reliability exercise (in English) involving both audio-taped interviews with study subjects and videotaped interviews with actors, role-playing a depressed subject. Item by item kappa statistics for SCAN items, were calculated comparing each interviewer's ratings against AF's "master" rating. A mean item by item kappa coefficient across all the sites of 0.77 (range 0.63 – 0.89) was obtained indicating a substantial level of inter-rater agreement.For inclusion in the first part of the linkage analysis, 944 affected subjects were genotyped from the 8 study sites as follows: Aarhus 48, Birmingham 146, Bonn 110, Cardiff 126, Dublin 154, Lausanne, 56, London 111 and St Louis 193. The age at interview and age of illness onset by gender of the subjects recruited at each site are shown in Table Mean age at interview for both sexes combined for each site were as follows: Aarhus 44.13 years (standard error of the mean (SEM)1.5), Birmingham 48.44 years (SEM 1.1), Bonn 51.46 years (SEM 1.2), Cardiff 44.06 years (SEM 0.9), Dublin 43.32 years (SEM 1.0), Lausanne 47.21 years (SEM 1.3), London 45.67 years (SEM 1.0), St Louis 47.14 years (SEM 0.9). These mean age differences were statistically significant : F = 6.26 degrees of freedom (df) 7, 936, p < 0.001. Tukey Post hoc test: Dublin, Cardiff, Aarhus, London, St Louis, Lausanne, Birmingham < St Louis, Lausanne, Birmingham, Bonn).Mean age at illness onset for both sexes combined per site were as follows: Aarhus 22.67 years (SEM 1.3), Birmingham 24.30 years (SEM 0.8), Bonn 27.28 years (SEM 1.2), Cardiff 23.47 years (SEM 0.9), Dublin 22.27 years (SEM 0.8), Lausanne 24.85 years (SEM 1.4), London 22.08 years (SEM 1.1), St Louis 18.17 years (SEM 0.8). These mean age differences were statistically significant (ANOVA: F = 9.82 df 7, 841 p < 0.001. Tukey Post hoc test: St Louis, London, Dublin, Aarhus, < London, Dublin, Aarhus Cardiff, Birmingham, Lausanne < Cardiff, Birmingham, Lausanne, Bonn).However, there were no significant differences between sites for the numbers of men and women recruited .Of the 944 subjects, 670 (71%) were female and 274 (29%) were males and hence, the female/male ratio was 2.45:1.Mean age at interview for all female subjects was 45.40 years (SEM 0.5) and for all males subjects was 45.69 (SEM 0.8). There were no significant gender differences for age at interview (t = -0.33, df = 477.58, p = ns)The mean age of illness onset for depressed male subjects was 22.61 years (SEM 0.7) compared to 22.52 years (SEM 0.4) for depressed female subjects. There was no significant sex difference for age of onset.(t = -0.11, df = 443.55 p = ns).Fifty five percent of male subjects and 61 % female subjects were living with a partner (married or cohabiting), while 45 % male subjects and 39% female subjects were living alone (ie separated, widowed, divorced or never married). Female subjects were significantly more likely to be living with a partner compared to male subjects. (Chi squared test = 26.89 df = 1 p < 0.001).There were 295 female and 117 male probands, 335 female and 144 male siblings and 40 female and 13 male parents included in the total sample. There were no significantly differences for the gender of probands, siblings or parents (chi squared test = 0.85, df = 2 p = ns).The mean age at interview for probands was 45.94 years (SEM 0.6) and for siblings was 45.80 years (SEM 0.5). There were no significant differences for age at interview between probands and their siblings (t = 0.17, df = 872.95 p = ns)Probands gave a mean age of illness onset of 20.22 years (SEM 0.6) while siblings reported a mean age of onset of 21.04 years (SEM 0.6). Again these differences were not statistically significant (t = -0.98, df = 882.93, p = ns)There were also no significant differences between probands and their siblings for marital status; 161 probands and 170 siblings were living alone while 242 probands and 290 sibings were living with a partner (chi squared test = 0.81 df = 1 p = ns).Genotyping was carried out by DeCode and the results checked for mis-specified relationships by the programs RELPAIR and Graphical Representation of Relationships (GRR) at the Institute of Psychiatry. RELPAIR compares the multipoint probability of the data conditional on the possible relationships, while GRR calculates the IBS mean and SD for each pair and plots these values, representing each type of relative pair using a different colour. Decisions about each problem pair were made on the basis of the results from both programs, although where there was discrepancy between the programs the GRR results were used.To check genotypes with Mendelian and other pedigree errors the PEDSTAT and MERLIN programs were used.These data cleaning processes resulted in 929 individuals being genotyped at 932 autosomal markers and 44 X chromosome markers. Success rates for the autosomal markers were above 61% and for 90% were above 86%. For the X chromosome the success rate was above 66%. For individuals the genotyping success rate was above 73% for autosomal markers and 61% for the X chromosome.The Depression Network study has recruited affected sibling pairs and some of their parents from 7 European and 1 North American site for a linkage analysis of recurrent unipolar depression. Because of differences in local service organisation, different recruitment strategies have been employed at the different sites. This may account for the significant differences for age at interview and age at illness onset between sites. The Bonn site recruited the oldest sibling pairs, both in terms of when subjects were interviewed and also when their illnesses had commenced. The Bonn subjects had a mean age at interview of 51.46 years compared to the Dublin subjects whose mean age at interview of 43.31 years was the youngest. Similarly the Bonn subjects mean age at illness onset was 27.28 years compared to a mean age of illness onset nearly a decade earlier for the St Louis subjects (18.17 years). It is noteworthy however that the St Louis sample included several large affected sibships. Subjects from families where there are many affected relatives may have a more genetic form of the disorder that might be contributing to an earlier age of onset.Despite these inter-site differences, the results show that there are also considerable similarities across the sites for the subjects recruited. Subjects have been mainly interviewed in their mid 40s and have experienced the onset of their recurrent depression in their early to mid 20s. Consequently subjects had on average around 20 years of history of episodes of depression when interviewed.As expected the study has shown the same preponderance of female to male subjects as many other studies with a gender ratio of around 2.45:1 [We would also not expect to find any significant differences between probands and siblings in terms of gender, age at interview, age at illness onset or marital status, which the results show is the case. Indeed for the purpose of finding genes for depression we would require siblings to have experienced similar forms of the illness.Although some subjects were excluded following genotyping due to errors that could not be reconciled, this preliminary genome linkage screen was able to include 929 subjects (98.4%) genotyped at 932 autosomal and 544 X chromosome markers. The results of the whole genome screen will be presented in due course.The Depression network study is the first co-ordinated international collaboration of its kind on the genetics of depression and one of the largest ever neuropsychiatric linkage study collection to use a uniform methodology to define and describe the phenotype. Despite taking place across eight sites and in six different countries good inter-rater agreement has been achievable as has good comparability of data.The study has been designed to overcome the difficulties that have been encountered in linkage studies of other psychiatric disorders such as schizophrenia and bipolar disorder. These started out optimistically with the assumption that genes of large effect would exist in at least some multiply affected families. However, after over a decade of contradictory findings and non replications, there is now consensus that such families are very rare or perhaps nonexistent. Rather it seems likely that common familial psychiatric disorders result from the combined effect of multiple genes none of which is either necessary or sufficient to cause the condition . ConsequThis study was funded by 3 year research grants to each participating site from Glaxo Wellcome Research and Development.AF & PMcG were overall study Principal Investigators (PIs), conceived the study and were co-ordinators of the study design, diagnostic reliability and data analysis. AF trained the interviewers from all the sites and wrote the paper. SB, LM and JP obtained the funding from Glaxo Wellcome, recruited site PIs and oversaw the quality of data collection, handling and analysis. GB analysed the genotyping data. The following authors were the individual site PIs in charge of all aspects of subject recruitment and data quality locally: OM Aarhus site, NC, LJ and IJ (joint) Birmingham site, MR and WM (joint) Bonn site, AK and MO (joint) Cardiff site, MG Dublin site, MP Lausanne site AF and PMcG (joint) London site and TR St Louis site. All authors have read and approved the contents of the final manuscript.The pre-publication history for this paper can be accessed here: