Chad A Galloway

John Ashton

Janet D Sparks

Robert A Mooney

Harold C Smith
email: harold.smith@rochester.edu

Metabolic regulation of APOBEC-1 Complementation Factor Trafficking in Mouse Models of Obesity and its Positive Correlation with the Expression of ApoB Protein in Hepatocytes

Keywords: diabetes, insulin-resistance, APOBEC-1 Complementation Factor, apolipoprotein B, mRNA editing ACF, APOBEC-1 Complementation Factor, ApoB, Apolipoprotein B, APOBEC-1, Apolipoprotein editing enzyme catalytic subunit 1, ELAV, embryonic lethal abnormal vision, ER, endoplasmic reticulum, GFP, green fluorescent protein, HDM, high density microsome, Il-6, interleukin 6, LDLR, low density lipoprotein receptor, LDM, low density microsome, NLS, nuclear localization sequence, NMD, nonsense mediated decay, PKC, protein kinase C, PP1, protein phosphatase 1, PPAR, peroxisome proliferationactivated receptor, RIA, radioimmunoassay, RNAi, RNA interference, RRM, RNA recognition motif, STAT, signal transducer

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Introduction

ACF (APOBEC-1 Complementation Factor) is a member of the ELAV/Hu family of RNA binding proteins, characterized by their multiple RNA Recognition Motifs (RRMs) [START_REF] Burd | Conserved structures and diversity of functions of RNA-binding proteins[END_REF]. This family of proteins functions in controlling the stability of AU-rich RNA [START_REF] Brennan | HuR and mRNA stability[END_REF]. ACF contains 3 RRMs in its N-terminus and has nanomolar affinity [START_REF] Mehta | Identification of Domains in APOBEC-1 Complementation Factor Required for RNA Binding and Apolipoprotein B mRNA editing[END_REF] for an 11 nucleotide AU-rich 'mooring' sequence 3' of the edited cytidine in apoB mRNA [4; 5]. ACF is a required factor for apoB mRNA editing site recognition, [6; 7] and recruitment of APOBEC-1, a cytidine deaminase, to nucleotide C6666 for site-specific C to U editing [START_REF] Teng | Molecular cloning of an apolipoprotein B messenger RNA editing protein[END_REF]. This RNA modification enables two functional protein products to be expressed from one gene [9; 10] (ApoB100 and ApoB48) that serve as scaffold proteins for lipoprotein particle assembly and transport of lipid from the liver and small intestine. Editing of apoB mRNA is a characteristic of differentiated tissue types [START_REF] Funahashi | Tissue-specific, developmental and nutritional regulation of the gene encoding the catalytic subunit of the rat apoB mRNA editing enzyme: functional role in the modulation of apoB mRNA editing[END_REF] and is regulated hormonally [START_REF] Mukhopadhyay | Thyroid hormone regulates hepatic triglyceride mobilization and apolipoprotein B messenger ribonucleic Acid editing in a murine model of congenital hypothyroidism[END_REF] and metabolically [13; 14; 15; 16; 17; 18]. While APOBEC-1 and apoB mRNA editing are not essential for life [START_REF] Nakamuta | Complete phenotypic characterization of the apobec-1 knockout mice with a wild-type genetic background and a human apolipoprotein B transgenic background, and restoration of apolipoprotein B mRNA editing by somatic gene transfer of Apobec-1[END_REF], ACF knockout was embryonically lethal [START_REF] Blanc | Targeted deletion of the murine apobec-1 complementation factor (acf) gene results in embryonic lethality[END_REF]. ACF was essential at the pre-implantation stage prior to the developmental induction of apoB mRNA and apoB mRNA editing [START_REF] Farese | Knockout of the mouse apolipoprotein B gene results in embryonic lethality in homozygotes and protection against dietinduced hypercholesterolemia in heterozygotes[END_REF], and therefore ACF must have had a biological function other than the complementation of apoB mRNA editing. In this regard, 'mooring' sequences have been identified in numerous mRNAs, some of which encode proteins involved in signal transduction and cell proliferation [START_REF] Grosjean | Fine-Tuning of RNA Functions by Modification and Editing[END_REF]. Whether there is an alternative function of ACF has been an open question based on the finding that it is abundantly expressed in human liver even though APOBEC-1 and editing activity are not observed in this tissue.

ApoB mRNA editing occurred in the cell nucleus on spliced polyadenylated message [23; 24]. Despite compartmentalization of apoB editing, ACF is distributed throughout the cell and was recovered from cell extracts as inactive 60S cytoplasmic complexes and active 27S nuclear editosomes [START_REF] Sowden | The editosome for cytidine to uridine mRNA editing has a native complexity of 27S: identification of intracellular domains containing active and inactive editing factors[END_REF]. ACF trafficking from the cytoplasm into the nucleus was dependent on an NLS (Nuclear Localization Sequence) in the C-terminal half of the protein [START_REF] Blanc | A novel nuclear localization signal in the auxiliary domain of apobec-1 complementation factor regulates nucleocytoplasmic import and shuttling[END_REF]. Increased nuclear abundance of ACF correlated with increased editing efficiency [12; 17; 27]. We recently reported that nuclear retention of ACF in response to metabolic stimulation correlated with phosphorylation of ACF's serine residue(s) by insulin-stimulated protein kinase C [27; 28]. It was also observed that nuclear accumulation of ACF resulted from the inhibition of Protein Phosphatase 1 (PP1), suggesting that ACF export required de-phosphorylation. These findings were of interest as immuno-electron microscopy of rat liver thin sections suggested that cytoplasmic ACF was localized to the exterior surface of the endoplasmic reticulum, the site of apoB translation [START_REF] Sowden | The editosome for cytidine to uridine mRNA editing has a native complexity of 27S: identification of intracellular domains containing active and inactive editing factors[END_REF]. The localization of ACF with apoB translational complexes also was supported by the finding that ACF was required to protect edited apoB mRNA from Nonsense Mediated Decay (NMD) [START_REF] Chester | The apolipoprotein B mRNA editing complex performs a multifunctional cycle and suppresses nonsense-mediated decay[END_REF]. This led us to the hypothesis that, in addition to mRNA editing, ACF may have a function in the export of apoB mRNA from the nucleus to the cytoplasm and its continued association with apoB mRNA may modulate the availability of apoB mRNA for ApoB protein translation. Evidence will be presented from studies in animal models for obesity that the abundance and trafficking of ACF were regulated processes that affected ApoB protein expression.

Materials and Methods

Animal care and primary hepatocyte preparation.

A C C E P T E D M A N U S C R I P T

Male B6.V-Lep ob /J, B6.Cg-m Lepr db /+ +/J and C57Bl/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME) and were used between 10 and 12 weeks of age, referred to herein as Ob/Ob, Db/Db, and cogenic lean control (respectively). Animals were housed in rooms with 12 hour light/dark cycles and fed normal rodent chow. Blood glucose levels were determined on blood drawn from the tail vein using a Bayer glucometer and Ascensia Contour Diabetic Test Strips. Fasting was conducted overnight for a 12 hour period. Insulin intraperitoneal injections were conducted with 1.5 IU/kg Novolin® N (Novo Nordisk, Princeton, NJ) and animals were sacrificed 4 hours following injection. Primary mouse hepatocytes were harvested as described previously [START_REF] Van Mater | Ethanol increases apolipoprotein B mRNA editing in rat primary hepatocytes and McArdle cells[END_REF]. Cells were plated in William's E Media (Sigma, St Louis, MO) with 5% FBS overnight prior to treatment on BIOCOAT type I collagen coated dishes (Becton Dickinson Labware).

Subcellular fractionation.

Whole liver extracts were prepared by Teflon to glass homogenization of liver in 0.25 STM (0.25 M sucrose, 50 mM Tris pH 7.0 and 5 mM MgCl 2 ) supplemented with Complete Protease Inhibitors (Roche, Indianapolis, IN) and phosphatase inhibitors, PhosStop (Roche) then cleared at 3,000 X g for 10 min. Nuclear and cytoplasmic preparations were centrifuged as previously described [START_REF] Smith | Analysis of protein complexes assembled on apolipoprotein B mRNA for mooring sequence-dependent RNA editing[END_REF] and the quality of the separation was assessed by western blotting extracts for cytoplasmic β-actin (Sigma) and nuclear histone H1 (Santa Cruz Biotechnology, Santa Cruz, CA). Sub-fractionation of cytoplasmic extract into HDM, LDM, and residual cytoplasmic fractions was conducted as previously described [START_REF] Phung | Phosphoinositide 3-kinase activity is necessary for insulin-dependent inhibition of apolipoprotein B secretion by rat hepatocytes and localizes to the endoplasmic reticulum[END_REF]. Subcellular fractionation of primary rat hepatocytes was conducted using the NE-PER kit (Thermo Scientific, Waltman, MA). Protein abundance in each fraction was assessed using the Bradford Assay (BioRad, Hercules, CA).

Immunological techniques and quantification.

A polyclonal antibody recognizing the C-terminus of ACF [START_REF] Lehmann | Metabolic regulation of apoB mRNA editing is associated with phosphorylation of APOBEC-1 complementation factor[END_REF] was used for the assessment of ACF abundance, while monoclonal antibodies recognizing either β-actin or histone H1 were used for normalization of protein input from the cytoplasmic or nuclear extracts (respectively). To assess total ACF, 40 µg of sample per lane were resolved on SDS-PAGE and western blots were sequentially probed for ACF and β-actin then visualized using ECL (Perkin Elmer Life Sciences, Waltham, MA). Nonsaturated film exposures were scanned and the intensity of signal quantified using ImageJ (NIH) software. Total ACF in each fraction was determined as a relative value by calculating the ratio of the whole cell ACF western blot signal to that of β-actin. The ACF/ β-actin ratio for control lean animals was set to 1 in order to evaluate changes in ACF abundance that may have occurred in the experimental conditions.

The calculations used in quantifying ACF nuclear and cytoplasmic distributions have been reported [START_REF] Sowden | Identification of Novel Alternative Splice Variants of APOBEC-1 Complementation Factor with Different Capacities to Support ApoB mRNA Editing[END_REF]. Briefly, to calculate the subcellular distribution of ACF, densitometric scans of ACF western blots of cytoplasmic and nuclear extracts were normalized for β-actin and histone H1 signals and mathematically corrected to total amount of protein recovered in each extract. These values were expressed as the nuclear to cytoplasmic ratio (Supplemental, Table S1). To assess the cytoplasmic and nuclear distribution of ACF protein was quantified using the BioRad assay kit (BioRad Laboratories, CA) and 120 µg or 40 µg of extract (respectively) were resolved by SDS-PAGE and probed sequentially for ACF, β-actin and H1. The ACF western signal was normalized by dividing the ACF signals by β-actin or histone H1signals in the cytoplasmic and nuclear lanes, respectively. A relative N:C ratio was then calculated by dividing the corrected ACF band density from the nuclear lane by the corrected ACF band density in the cytoplasmic lane. The fold-change in ACF N:C ratio from that seen in the control animals was calculated by dividing the N:C ratio calculated for the insulin treated animal by the N:C ratio of the saline treated animal. Reported averages are from two trials.

ApoB Quantification.

For western blotting, equivalent µg amounts of cytoplasmic extract from Ob/Ob and C57bl/6J mice were probed with an antibody recognizing the N-terminus of ApoB (Santa Cruz Biotechnology, Santa Cruz, CA) to assess the relative abundance of intracellular ApoB100 and ApoB48 proteins. For radioimmunoassay (RIA) assessment of ApoB, media was replaced at 24 postinfection of rat primary hepatocytes infected with virus expressing RNAi against acf. Media was harvested 24 hours following replacement and assessed by a quantitative RIA using a mouse monoclonal antibody raised against the N-terminus of rat ApoB [START_REF] Sparks | The production and utility of monoclonal antibodies to rat apolipoprotein B lipoproteins[END_REF]. ApoB secretion was calibrated using a standard curve prepared from rat VLDL. In addition, ApoB secreted in the media, as well as intracellular ApoB in whole cell lysates was quantified following 5 hr drug treatment of primary hepatocytes with at 5 µM Cantharidin. Cellular and media ApoB were normalized to whole cell protein content and results were reported as ng ApoB/mg cell protein as described above.

In vivo phosphorylation.

In vivo 32 Pi labeling was performed by intraperitoneal injection of mice with 0.2 mCi/g BW of orthophosphoric acid (NEN, Boston, MA) buffered with 50 mM HEPES, pH 7.0 and 150 mM NaCl. After 4 h, mice were sacrificed and hepatic cytoplasmic and nuclear extracts prepared as described above.

RNA analyses.

Total hepatic abundance of editosome component (acf, apoB and apobec-1) mRNAs in each mouse model was determined, using RNA isolated from 100 mg of fresh tissue homogenized in TRI-Reagent (MRC, Cincinnati, OH) as per manufacturer's instructions. cDNA was prepared by amplification of 2 µg of RNA primed by oligo dT and amplified using real time PCR. Real time reactions were monitored using TaqMan® (ABI, Foster City, CA) master mix with Exiqon mouse probes 60, 40, 59, 42, 26, 69 and 43 for apobec-1, apoB, acf, cugbp2, hnrnpa1, gryrbp and β-actin, respectively, and using the following primers: apobec- For evaluating the effect of hormone or metabolic treatment on the abundance of mRNAs encoding editosome components, 60 mm dishes of primary hepatocytes were rinsed twice in 1X PBS then harvested by scraping into TRI-Reagent (MRC). Two µg of RNA were used to amplify cDNA input for real time PCR. Subcellular fractionation of whole liver was carried out as described above with equivalent portions of cytoplasmic and nuclear extract placed in TRI-Reagent (MRC) for assessment of compartmental apoB abundance in Ob/Ob mice. Subcellular fractionation of primary rat hepatocytes following cantharidin treatment was carried out as described above. Total RNA recovered from the extraction was poly A purified using MicroPoly (A) Pursuit (Ambion, Austin, TX) and 150 ng were used as input for cDNA reaction to be used for real time PCR. β-actin was used to normalize for input in real time PCRs.

ApoB mRNA editing was quantified by the poisoned primer extension assay as previously described [START_REF] Backus | Quantitation of endogenous liver apolipoprotein B mRNA editing[END_REF] on apoB cDNA amplified from 2 µg of total RNA isolated from the liver of Ob/Ob and C57Bl/6J mice as described above.

Treatments of primary hepatocytes.

All hepatocytes were prepared as previously described and allowed to equilibrate overnight in fresh media [START_REF] Van Mater | Ethanol increases apolipoprotein B mRNA editing in rat primary hepatocytes and McArdle cells[END_REF]. Hormones used to treat mouse primary hepatocytes were: leptin, TNFα, IL-6, and resistin, were purchased from R&D systems (Minneapolis, MN) and the doses used in each treatment were based on recommended EC 50 concentrations by the manufacturer: 0.01 ng/mL, 0.05 ng/mL, 0.02 ng/mL and 10 µg/mL, respectively. Hourly treatments for 4 hours at the suggested EC 50 , as well as 10-fold above and below the EC 50 were tested. Resistin was only used at the provided EC 50 . Equal volumes of PBS were added as control treatments. Free fatty acid treatment was carried out for a period of 4 hours at two concentrations, 0.1 and 1 mM using a 2:1 ratio of oleate to palmitate [START_REF] Gomez-Lechon | A human hepatocellular in vitro model to investigate steatosis[END_REF].

RNAi Constructs and

Treatment. Three non-conserved 22 nucleotide sequences were selected as targets of acf message, GenBank Accession Number NM_133400.1, using the RNAi Codex server at http://codex.cshl.edu.

Perspective targets were PCR amplified from oligos; H2, TGCTGTTGACAGTGAGCGCGTTGTTGATGTCATTGTCTACTAGTGAAGCCACAGATGTAGTA GACAATGACATCAACAACTTGCCTACTGCCTCGGA, H3, TGCTGTTGACAGTGAGC GCACAGAGGCTATGCATTTGTAATAGTGAAGCCACAGATGTATTACAAATGCATAGCCTCT GTTTGCCTACTGCCTCGGA and W2, TGCTGTTGACAGTGAGCGAAATCCTGTAC GTAAGGAACCTTTAGTGAAGCCACAGATGTAAAGGTTCCTTACGTACAGGATTTTGCCTACT GCCTCGGA containing the selected targets and mir-30 sequence context, including the hairpin loop structure, as previously described [START_REF] Paddison | Short hairpin activated gene silencing in mammalian cells[END_REF] using the primers 5'CAGAAGGGGATCCAAGGTATA TTGCTGTTGACAGTGAGCG and 3' CTAAAGTAGCCCCTTAAGCTTCCGAGGCAGTAGGCA. A nonspecific control sequence NS, TGCTGTTGACAGTGAGCGATCTC GCTTGGGCGAGAGTAAGTAGTGAAGCCACAGATGTACTTACTCTCGCCCAAGCGAGATTGCC TACTGCCTCGGA also was amplified. The resulting PCR products were cloned individually into pAd shRNAmU6 vector [START_REF] Ro | Adenovirus-based short hairpin RNA vectors containing an EGFP marker and mouse U6, human H1, or human U6 promoter[END_REF] using BamHI and HindIII restriction sites. Resulting vectors were recombined with pAdEasy-1 in the BJ5183-AD-1 E. coli strain and infective virus produced as per manufacturer's instructions using the pAdEasy XL Adenoviral Vector System (Stratagene, La Jolla, CA). McArdle cells at 80% confluence were treated with 10 µL of virus for 2 hours in minimal media before replacing the media. Infectivity was monitored by fluorescence microscopy 24 hrs post infection prior to preparing whole cell lysates using Reporter Lysis Buffer (Promega, Madison, WI) per manufacture protocol. Primary rat hepatocytes were plated as previously described, treated with 30 µL of virus for 2 hrs in minimal media and then incubated with fresh media. Infectivity and harvest were conducted as described for McArdle cells at 48 hours post infection.

Results

Dysregulation of ACF in the genetically obese and insulin-resistant Ob/Ob mouse.

Given that the normal hepatic response to insulin was to retain ACF in the nucleus, we evaluated whether this response was retained in insulin-resistant mice. We examined this question in the leptin-deficient Ob/Ob mice, a model for obesity-induced insulin-resistance. In Ob/Ob mice, hyperphagia, due to a lack of functional leptin, begins from birth and is responsible for the obese phenotype and leads to insulinresistance in adult animals [START_REF] Zhang | Positional cloning of the mouse obese gene and its human homologue[END_REF]. Ob/Ob mice were determined to be obese and diabetic, averaging twice the body weight of cogenic lean controls (40.1g vs. 21.4g) and characteristic of insulin-resistance, these mice had blood glucose levels approximately 2-fold higher than observed in lean mice (372 + 70 mg/dL vs. 182 + 30 mg/dL).

Using real-time-PCR, we examined the expression of hepatic acf, apobec-1 and apoB mRNAs using β-actin mRNA as a normalization control. While both apobec-1 and apoB mRNA appeared to be slightly more abundant in Ob/Ob mouse liver relative to cogenic lean control mice (normalized to β-actin mRNA abundance), these changes were not statistically significant with p≤0.29 and p≤0.17, respectively (Fig. 1A). Unexpectedly, the abundance of acf message was increased nearly 7-fold (p≤0.05) in Ob/Ob mouse liver. By western blotting, Ob/Ob total hepatic ACF protein abundance was increased approximately 5-fold (p≤0.01) relative to the abundance of hepatic ACF protein in lean control mice (normalized to β-actin protein abundance) (Fig. 1B). Previous studies showed that CUGBP2 [12; 38] and GRYRBP [39; 40] and hnRNPA1 [START_REF] Blanc | Identification of GRY-RBP as an apolipoprotein B RNA-binding protein that interacts with both apobec-1 and apobec-1 complementation factor to modulate C to U editing[END_REF] are RNA-binding proteins that traffic between the nucleus and cytoplasm and whose interactions with apoB mRNA and ACF inhibit apoB mRNA editing. We evaluated whether the mRNAs encoding these proteins also were up regulated in Ob/Ob mouse liver. Real-time PCR showed that while cugbp2 mRNA was slightly elevated (1.7-fold, p<0.4) relative to that quantified in lean controls, the abundance of hnrnpa1 and gryrbp mRNAs were not altered (Supplemental Fig S1). The data suggested that there was a significant difference in the magnitude of up regulation seen with ACF in Ob/Ob liver compared with other RNA binding proteins we evaluated. We therefore focused on ACF for the remainder of this study.

To determine what was responsible for the elevated expression of ACF mRNA and protein, we considered that the Ob/Ob mice lacked leptin and that this may have indirectly or directly contributed to our findings. In addition to leptin however, obesity is associated with increased levels of other hormones such as the adipokine resistin and pro-inflammatory cytokines Il-6 and TNFα [41; 42; 43]. To evaluate the effect of these factors on acf mRNA expression under defined in vitro conditions, primary hepatocytes from lean mice were treated with either resistin, Il-6, TNFα or free fatty acids [34; 44]. All of these treatments failed to modulate acf mRNA abundance (data not shown). However, treatment of primary hepatocytes with leptin demonstrated a dose-dependent suppression of acf mRNA (p≤0.05) (Fig. 2). Consequently, the ability of leptin to alter acf mRNA abundance was unique to the agents we tested. Although ACF protein levels were not significantly suppressed by leptin during this 4 hour time course (data not shown), our findings are consistent with the possibility that the absence of leptin during the lifespan of the Ob/Ob mouse contributed to the over expression of ACF. However, these data did not rule out the possibility that insulin resistance in the Ob/Ob mouse liver also may have contributed to the observed changes in ACF abundance.

To further explore the potential suppressive effects of leptin on acf mRNA we quantified acf, apoB and apobec-1 mRNAs in the livers from the Db/Db genetic mouse model for obesity-induced insulin resistance. In these mice, leptin is expressed, but knockout of the long form of the leptin receptor (Ob-Rb), expressed primarily in the brain, induces hyperphagia leading to obesity and insulin resistance. Important to this study, despite knockout of Ob-Rb, leptin signaling in the liver is intact due to a short form of the leptin receptor (Ob-Ra) expressed in the liver [45; 46]. Both apoB and apobec-1 mRNAs were suppressed in Db/Db liver to statistically significant levels relative to that in the livers of cogenic littermates (p≤0.01 and p≤0.05, respectively). However, acf mRNA levels were not significantly different from that seen in control livers (Supplemental Fig. S2, left panel). Total ACF protein levels in the Db/Db mouse liver also were not significantly different from that quantified in control animals (Supplemental Fig. S2, right panel). The data suggested that leptin signaling in Db/Db and lean control mouse liver maintained comparable levels of ACF mRNA and protein expression. Taken together, these findings supported a novel role for leptin in regulating hepatic ACF abundance but that insulin resistance did not make a significant contribution to the regulation of ACF abundance.

ACF trafficking defect in Ob/Ob mouse liver.

Given the large increase in ACF expression in the liver of Ob/Ob mice together with their insulin resistance, it was unclear whether these animals retained the capacity for insulin-dependent regulation of ACF nuclear retention. Prior to liver harvest and subcellular fractionation, animals were fasted and received either control saline or 1.5 IU insulin/kg body weight by intraperitoneal injection to promote ACF's nuclear retention. Consistent with previous published findings in rats [START_REF] Sowden | The editosome for cytidine to uridine mRNA editing has a native complexity of 27S: identification of intracellular domains containing active and inactive editing factors[END_REF], fasting of control mice resulted in a marked increase in the proportion of total cellular ACF in the cytoplasm and insulin treatment produced a 4-fold increase in nuclear ACF abundance within 4 hours following injection (Fig. 3A and relevant quantification in Supplemental Table S1). The calculated proportion of ACF that traffics in response to insulin represents only a small fraction of the total cellular ACF. Nonetheless, these data demonstrated that the previously characterized insulin-dependent nuclear retention of ACF in rats [17; 25] was conserved in mice.

In contrast to the lean control mice, Ob/Ob hepatic ACF was not responsive to fasting. ACF failed to redistribute to the cytoplasm (Fig. 3A). Moreover, insulin administration did not promote nuclear accumulation of ACF in Ob/Ob mouse liver as evidenced by the calculated 'no difference' (1-fold) in the relative nuclear to cytoplasmic ratio of ACF in insulin-treated versus saline-treated animals (Fig. 3A). Similar analyses were conducted using Db/Db animals to evaluate whether these animals also lacked an insulin response in ACF trafficking in the absence of the ACF over expression observed in Ob/Ob mice. Insulin injection did not stimulate nuclear retention of ACF in Db/Db mice (Fig. 3B) suggesting that the lack of a robust response was a general characteristic of the obesity-induced insulin-resistance state and was not simply due to ACF over expression characteristic of Ob/Ob mice.

ApoB mRNA Editing is resistant to insulin stimulation in Ob/Ob liver.

Our laboratory [17; 25] and others [15; 47] have shown that apoB mRNA editing is stimulated by insulin. Given that Ob/Ob mice are hyperinsulinemic [START_REF] Baetens | Alteration of islet cell populations in spontaneously diabetic mice[END_REF] we had anticipated a greater amount of nuclear retention of hepatic ACF. Because this was not observed, we questioned whether editing activity itself had become insulin-resistant. The proportion of total hepatic apoB mRNA edited in fed Ob/Ob and Db/Db mice was quantified by the poisoned primer extension assay (Fig. 4A). Ob/Ob and Db/Db liver editing was 67% +/-3% (n=3) and 56% +/-5% (n=3) respectively and therefore lower than that quantified in fed lean control animals 73% +/-1%. These findings suggested that apoB mRNA editing was not responsive to the elevated insulin levels that were present in fed obese mice.

We have previously shown that elevated insulin levels correlated with an enhanced abundance of phosphoACF in the nucleus of hepatocytes [START_REF] Lehmann | Metabolic regulation of apoB mRNA editing is associated with phosphorylation of APOBEC-1 complementation factor[END_REF]. To evaluate whether Ob/Ob hepatocytes retained the ability to phosphorylated ACF, hepatic extracts were prepared from fed control and Ob/Ob mice that had been injected intraperitoneally with 32 Pi. Nuclear and cytoplasmic extracts were immunoprecipitated with the C-terminal ACF polyclonal antibody and the ACF recovered analyzed by SDS PAGE and autoradiographed. ACF was determined to be phosphorylated and phosphoACF was properly compartmentalized to the nucleus in both lean controls and Ob/Ob mouse livers (Fig. 4B). Quantification of the 32 P signal recovered with nuclear ACF per µg of protein suggested that Ob/Ob nuclear ACF had twice the specific labeling of ACF compared to lean control ACF (Fig. 4C). Although phosphorylation of ACF is not required for apoB mRNA binding [27; 28], the apparent stimulation of nuclear ACF phosphorylation without a commensurate stimulation in apoB mRNA editing suggested some insulin regulation of protein kinase C activity on ACF remained intact but that editosome assembly and/or activity was insulin-resistant in the Ob/Ob hepatocyte. The data suggest an uncoupling of the previously observed role of ACF phosphorylation in the stimulation of apoB mRNA editing.

Increased cytoplasmic ACF correlates with increased cytoplasmic apoB mRNA and enhanced cytoplasmic ApoB protein.

Given the localization of cytoplasmic ACF to the exterior surface of the endoplasmic reticulum (ER), enhanced ACF expression may have facilitated apoB mRNA translation. To evaluate whether the enhanced cytoplasmic levels of ACF had an impact on apoB mRNA, poly A+ mRNA was isolated from nuclear and cytoplasmic extracts of Ob/Ob and C57Bl/J6 mice and apoB and β-actin sequences amplified by real time PCR amplification. Despite the finding that total hepatic cellular apoB mRNA was not significantly increased in Ob/Ob mice relative to lean control mice (Fig. 1), a 2.5-fold increase (p≤0.01) in cytoplasmic apoB mRNA abundance was observed in Ob/Ob mice relative to that quantified in the cytoplasm of lean controls (Fig. 5A). The result was commensurate with a reduction in the relative abundance of nuclear apoB mRNA (p≤0.01) in Ob/Ob animals (Fig. 5A). Interestingly, within hepatic cytoplasmic extracts of both mice and rats, ACF was localized to the low density microsomal fraction (LDM) (Supplementary Fig. S3). This cytoplasmic fraction in rat hepatocytes has been described to contain approximately 75% of cytoplasmic apoB message and apoB translation complexes [START_REF] Phung | Phosphoinositide 3-kinase activity is necessary for insulin-dependent inhibition of apolipoprotein B secretion by rat hepatocytes and localizes to the endoplasmic reticulum[END_REF]. Cytoplasmic ACF in the Ob/Ob mice was recovered in both LDM and high density microsomal (HDM) fractions suggesting that a preponderance of ACF that is over expressed in Ob/Ob mice may retain its association with apoB mRNA in the cytoplasm.

From these data we predicted that elevated ACF in the LDM fraction in Ob/Ob livers may stabilize apoB mRNA translation complexes associated with the ER. Moreover, the expression of ApoB48 from edited apoB mRNA might be enhanced due to the ability of the additional ACF to more effectively protect edited mRNA from NMD [START_REF] Chester | The apolipoprotein B mRNA editing complex performs a multifunctional cycle and suppresses nonsense-mediated decay[END_REF]. Analysis of the western blots of cytoplasmic fractions revealed a 60% increase in total ApoB (p≤0.07) (Fig. 5D) with the majority of the increase in the abundance of ApoB48 (increased 2-fold in Ob/Ob animal p≤0.01) (Fig. 5C). These findings are strong support for our hypothesis that alterations in the regulation of ACF can affect downstream pathways involved in ApoB protein expression.

ACF Localization Affects ApoB Secretion.

Our hypothesis predicts that nuclear export of ACF could affect apoB mRNA nuclear export to the cytoplasm and its translation. To further assess this prediction we returned to our previously characterized nuclear retention of ACF in rat primary hepatocytes treated with cantharidin to inhibit PP1 [START_REF] Lehmann | Metabolic regulation of apoB mRNA editing is associated with phosphorylation of APOBEC-1 complementation factor[END_REF]. Cantharidin treatments of primary rat hepatocytes at 5 µM, 10X the IC50 of PP1, resulted in a marked increase in nuclear ACF, coordinate with a reduction of cytoplasmic ACF (Fig. 6A). In parallel, intracellular and secreted ApoB protein were assessed by radioimmunoassay [START_REF] Sparks | The production and utility of monoclonal antibodies to rat apolipoprotein B lipoproteins[END_REF] and normalized for protein input. A reduction in both intracellular (p≤0.02) and secreted (p≤0.001) ApoB was observed relative to DMSO control treatment (Fig. 6B). While the most robust inhibition was observed in ApoB secretion, the reduction of intracellular ApoB was also significant. The data suggested a requirement for PP1 activity for proper packaging and secretion of nascent ApoB VLDL as well as possibility that PP1 activity may have been requirement for the translation or stability of ApoB itself. Real time PCR demonstrated that while apoB mRNA was slightly elevated in the cytoplasmic fraction of cantharidin treated hepatocytes the result was not statistically significant (p≤0.32) (Fig. 6C). However, the ~2-fold increase in the nuclear abundance of apoB mRNA was statistically significant (p≤0.05). These findings supported our hypothesis that ACF nuclear export was consequential for the amount of ApoB protein that was expressed and secreted however we cannot discount from this experiment that cantharidin treatment may have affected other processes in hepatocytes that were also responsible for the reduction in ApoB secretion.

ACF Abundance Affects ApoB Secretion.

To demonstrate that ApoB expression was indeed modulated by ACF availability and abundance, an RNAi knockdown analysis was conducted in rat primary hepatocytes. Constructs were developed in the pAdEasy system to target rat acf mRNA. Each of three adenoviruses targeting one of 3 unique sites within acf mRNA were assessed in McArdle cells, a rat hepatic cell line, for their efficacy along with virus encoding a nonspecific and random sequence as a control 'NS' (Fig. 7A). The target site designated 'H2' resulted in the most efficient reduction of ACF protein and was subsequently used in treatment of primary rat hepatocytes where it demonstrated an MOI-dependent reduction of ACF protein (Fig. 7B). NS was without effect on ACF abundance. Infectivity was nearly 100% at the maximal MOI as assessed by fluorescence microscopy visualizing GFP expression expressed off a CMV promoter 3' to the shRNAi (data not shown). The abundance of secreted ApoB protein was evaluated by RIA in media harvested 48 hours following infection of primary hepatocyte cultures. H2 expression induced approximately 40% reduction of secreted ApoB protein compared to cells expressing NS (Fig. 7C). These findings are significant in light of the foregoing experiments as they underscore the possibility that hepatic ACF participates at an early stage in the regulation of apoB mRNA availability for translation in the cytoplasm.

Discussion

In this report we describe a novel role for ACF in the regulation of apoB mRNA export from the nucleus and the importance of this process in animal models for modulating the expression of ApoB protein. The function of ACF to date has been limited to its role as an auxiliary factor required for apoB mRNA editing in mammalian small intestine and in the liver of many species [START_REF] Greeve | Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoBcontaining plasma lipoproteins[END_REF]. Our new findings provide an intriguing potential hypothesis for why ACF expression has been retained in human and nonhuman primate livers despite the loss of APOBEC-1 expression and lack of apoB mRNA editing activity [START_REF] Greeve | Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoBcontaining plasma lipoproteins[END_REF].

An important implication of the findings from this study was that ACF may function early in the regulatory pathways that control lipoprotein synthesis. Prior to our study, research on ACF focused on its role in apoB mRNA editing [START_REF] Smith | Measuring editing activity and identifying cytidine-to-uridine mRNA editing factors in cells and biochemical isolates[END_REF] and studies of ApoB focused on lipoprotein assembly and secretion [START_REF] Gibbons | Synthesis and function of hepatic very-lowdensity lipoprotein[END_REF]. Early work from this lab showed that ACF trafficking was regulated by insulin-dependent serine phosphorylation [27; 28]. We reasoned that Ob/Ob mice might enable an evaluation of ACF trafficking and ApoB expression in the context of an insulin-resistant disease model. We observed that the levels of hepatic acf message and protein were markedly increased. This change stood out for the magnitude of change in acf mRNA levels relative to that of related RNA binding proteins.

The progression to obesity in Ob/Ob mice begins immediately after birth due to hyperphagia. This manifests in an increased abundance of adipose tissue, altered response to insulin stimulation and exposure of the liver to increased levels of serum insulin, adipokines (leptin [START_REF] Uygun | Serum leptin levels in patients with nonalcoholic steatohepatitis[END_REF] and resistin [START_REF] Yagmur | Resistin serum levels are associated with insulin resistance, disease severity, clinical complications, and prognosis in patients with chronic liver diseases[END_REF]), proinflammatory cytokines (Il-6 and TNFα [START_REF] Tilg | Adipocytokines: mediators linking adipose tissue, inflammation and immunity[END_REF]), and free fatty acid flux [START_REF] Araya | Increase in long-chain polyunsaturated fatty acid n -6/n -3 ratio in relation to hepatic steatosis in patients with non-alcoholic fatty liver disease[END_REF]. In testing several of these factors individually, under the defined conditions of primary hepatocyte culture, we found that only leptin had the ability to alter acf mRNA abundance, causing its suppression in a dose-dependent manner. Supporting a role for leptin in ACF expression was the finding that insulin resistance and obesity were not sufficient to change ACF expression in the Db/Db mice that retained leptin signaling in their livers.

The regulation of ACF abundance by leptin is only the second example of hormonal regulation of acf message. Previously, thyroid hormone was shown to increase the abundance of ACF in neonatal Pax8 -/-mice [START_REF] Mukhopadhyay | Thyroid hormone regulates hepatic triglyceride mobilization and apolipoprotein B messenger ribonucleic Acid editing in a murine model of congenital hypothyroidism[END_REF]. Treatment with thyroid hormone resulted in a mobilization of hepatic triglycerides, which the authors hypothesized was due to increases in the proportion of ApoB48 availability [START_REF] Mukhopadhyay | Thyroid hormone regulates hepatic triglyceride mobilization and apolipoprotein B messenger ribonucleic Acid editing in a murine model of congenital hypothyroidism[END_REF]. It is of interest that the PPARα agonist, ciprofibrate, also reduced ACF abundance in Low Density Lipoprotein

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Receptor (LDLR) knockout animals [START_REF] Fu | The peroxisome proliferator-activated receptor alpha (PPARalpha) agonist ciprofibrate inhibits apolipoprotein B mRNA editing in low density lipoprotein receptor-deficient mice: effects on plasma lipoproteins and the development of atherosclerotic lesions[END_REF]. In these studies, the amount of edited apoB mRNA varied in direct proportion with ACF abundance. In contrast, we observed no significant changes in the basal level of apoB mRNA editing in Ob/Ob mice relative to that of the lean control. These findings together with the data showing that Ob/Ob hepatocytes failed to up-regulate ACF retention in the nucleus underscore the importance of insulin for controlling the function of ACF in the lean physiological state.

Our studies employed two animal models for obesity. Ob/Ob mice express leptin receptors, but do not express leptin and consequently become hyperphagic and obese. Db/Db mice express leptin but lack the leptin receptor in their CNS and become hyperphagic and obese. Obesity led to insulin resistance in both animal models. We found that hepatic ACF nuclear retention and apoB mRNA editing activity were insulin-resistant in both animal models. Db/Db hepatic acf mRNA and ACF protein abundances were not elevated above control whereas Ob/Ob acf mRNA and ACF protein levels were markedly elevated. We attribute this difference to the expression of leptin and leptin signal in Db/Db liver but not in the Ob/Ob liver [START_REF] Chen | Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice[END_REF]. There are several splice variants of the leptin receptor that differ in the length of their cytoplasmic domain required for docking of Janus kinase and STAT proteins, through which leptin signals [53; 54]. Signaling through the long form of leptin receptor (Ob-Rb) is unlikely to have contributed to acf mRNA suppression as only the truncated leptin receptor (Ob-Ra) is expressed [START_REF] Sahai | Obese and diabetic db/db mice develop marked liver fibrosis in a model of nonalcoholic steatohepatitis: role of short-form leptin receptors and osteopontin[END_REF]. The Ob-Ra receptor lacks the consensus box 1 and 2 motifs utilized for docking of Janus kinase and therefore its signaling does not go through a STAT3 dependent pathway [START_REF] Yamashita | Leptin receptor signal transduction: OBRa and OBRb of fa type[END_REF]. Signaling through the Ob-Ra receptor has been described through MAPK activation [START_REF] Yamashita | Leptin receptor signal transduction: OBRa and OBRb of fa type[END_REF]. Functional Ob-Ra receptor signaling has been shown through induced expression of c-fos, c-jun, and jun-B [START_REF] Murakami | A short form of leptin receptor performs signal transduction[END_REF]. Leptin signaling in the Db/Db liver may have remained responsive and therefore continued to the suppression of acf mRNA abundance. The regulatory factors that normally modulate ACF expression will be an important area for future research.

We can not rule out that over expression of ACF may have compounded the defect in regulating ACF and apoB mRNA nuclear export and ApoB translation in the Ob/Ob mouse but our data suggested that insulin resistance was the primary reason for why ACF nuclear retention and apoB mRNA editing activity were not elevated above the lean control values as would have been anticipated given the hyperinsulinemic condition in these animals. Considering that ACF was hyper-phosphorylated on serine residues in Ob/Ob liver and phosphoACF was restricted in the nucleus, PKC activity on nuclear ACF and factors determining nuclear retention must have retained a basal level of function. However, the possibility remains that insulin resistance involved reduced activation of PKC and/or heightened protein phosphatase I activity. This might explain why the radioactivity per µg of ACF protein in Ob/Ob nuclei was only 2-fold higher than that observed in the lean control when an earlier study showed that nuclear ACF specific radioactivity increased approximately 5-fold in lean mice following a 10 nm insulin injection [START_REF] Lehmann | Metabolic regulation of apoB mRNA editing is associated with phosphorylation of APOBEC-1 complementation factor[END_REF].

An equally plausible explanation for our findings is that nuclear protein kinase C activity may have become insufficient for the level of ACF phosphorylate necessary to affect nuclear restriction because this pathway was less responsive to insulin. Given that apoB mRNA editing efficiency was suppressed in Ob/Ob and Db/Db mouse liver compared to lean control liver, our study suggested that the insulin-dependent processes that otherwise regulate ACF assembly into editosomes may have been compromised by the inability to restrict adequate amounts of ACF to the nucleus.

In published studies and in this study, the bulk of total cellular ACF was recovered with cytoplasmic extracts from whole liver or hepatocytes [17; 25]. Immuno electron microscopy showed that cytoplasmic ACF was predominantly localized along the exterior of the endoplasmic reticulum [START_REF] Sowden | The editosome for cytidine to uridine mRNA editing has a native complexity of 27S: identification of intracellular domains containing active and inactive editing factors[END_REF]. This is also the intracellular site of apoB mRNA translation where microsomal triglyceride transfer protein facilitates co-translational lipid loading onto nascent ApoB [57; 58]. Differential centrifugation of liver cytoplasmic extracts yielded microsomal fractions in which the rough endoplasmic reticulum (high density microsomal fraction, HDM), smooth endoplasmic reticulum and Golgi stacks (low density microsomal fraction, LDM) and residual cytoplasm were separated [START_REF] Graham | Fractionation of a microsomal fraction from rat liver or cultured cells[END_REF]. The bulk of nascent ApoB protein co-fractionated with the LDM fraction [START_REF] Phung | Phosphoinositide 3-kinase activity is necessary for insulin-dependent inhibition of apolipoprotein B secretion by rat hepatocytes and localizes to the endoplasmic reticulum[END_REF]. Using this protocol, we found that most of the cytoplasmic ACF was not recovered as a soluble protein with the residual cytoplasm fraction but rather, was enriched in the LDM and HDM factions. This characteristic of ACF was conserved in mice and rats. In fact, despite the high level of ACF expression in Ob/Ob mouse liver, the association of ACF with microsomes was maintained. The relative recovery of ACF with the Ob/Ob HDM fraction compared with the LDM fraction, suggested that there was more ACF in the HDM fraction of obese mice compared with HDM of lean control mice. This association is consistent with the hypothesis that ACF maintained it association with apoB mRNA following nuclear export to the sites of ApoB protein synthesis in the cytoplasm. The importance of ACF abundance and trafficking for ApoB production was underscored by the finding that ApoB secretion was inhibited through RNAi knockdown of ACF. An additional correlation between ACF trafficking and ApoB synthesis and secretion was that nuclear retention of ACF through PP1 inhibition was associated with reduced cytoplasmic apoB mRNA and increased nuclear apoB mRNA in primary hepatocytes. Consistent with our hypothesis, intracellular and secreted ApoB protein was reduced in primary hepatocytes treated with Cantharidin. We propose therefore that the inability of the liver in obese animals to appropriately regulate nuclear retention of ACF is what led to dysregulation of ApoB.

More detailed analysis will be necessary to de-convolute the regulation of ACF trafficking in the insulin-resistant state. Future studies will focus on developing genetic models to determine the role of ACF nuclear and cytoplasmic trafficking in mRNA export from the nucleus and identify the signaling pathways that regulate ACF expression and post-translational modification. Specifically, this study has demonstrated using animal models a role for leptin signaling in the regulation of ACF expression and shown that deficiencies in nuclear retention of ACF was part of the obesity-induced, insulin-resistant phenotype. Importantly our studies suggested that independent of the function ACF has in apoB mRNA editing activity (resulting from its interaction with APOBEC-1), ACF may have a primary function as an apoB mRNA recognition factor and chaperone. This function suggested a new hypothesis that may explain why ACF continues to be expressed in nonhuman primate and human livers even though apoB mRNA editing activity is no longer possible. It is also possible that ACF serves to chaperone other mRNAs that are important for development and it is the loss of this function that accounts for why ACF knockouts were not viable. It will be important to develop a conditional ACF knockout animal model to address this possibility. Subcellular fractionation was performed to generate nuclear and cytoplasmic extracts of which 40 and 120 µg (respectively) were resolved by SDS-PAGE and transferred to nitrocellulose. Western blots were probed with ACF peptide-specific antibody and for β-actin and histone H1 as fraction-specific loading controls for the cytoplasm and nucleus (respectively). The nuclear to cytoplasmic ratio (N:C) of ACF signal was calculated as described in Materials and Methods and Supplemental Table 1. The fold change in N:C ratio was calculated (as the relative N:C ratio insulin-treated mice divided by the relative N:C ratio measured in saline-treated mice). The calculations are the average of two trials.

Figure. 4.

ACF phosphorylation and apoB editing are uncoupled in Ob/Ob mice. A. ApoB mRNA was amplified from 2 µg of total RNA and apoB mRNA editing efficiency was evaluated by poisoned primer extension as previously described [START_REF] Smith | Measuring editing activity and identifying cytidine-to-uridine mRNA editing factors in cells and biochemical isolates[END_REF]. Shown is the editing activity of in the liver of three different animals. The percent editing activity is the average of the editing activity determined in each animal and was calculated as a quotient of the signal from the primer extension product of edited apoB mRNA divided by the signals from primer extension products from the edited and unedited mRNAs multiplied by 100. B. Fed mice were injected with 32 Pi buffered orthophosphoric acid 4 hours prior to liver harvest and subcellular fractionation. Cytoplasmic and nuclear extracts were immunoprecipitated with ACF Cterminal specific polyclonal antibody, resolved by SDS PAGE, transferred to PVDF membrane and autoradiographed. C. The specific activity of ACF was calculated as the PhosphorImager quantified 32 P signal divided by ACF western signal. The calculated specific activity and error are the average of a determination performed on an extract from each of three animals. The relative nuclear and cytoplasmic abundance of apoB mRNA in control mice was set to one. The statistical significance was determined to be p≤0.01 for both cytoplasmic and nuclear differences using a student's t-test with n=3. B. Hepatic cytoplasmic extracts from 3 different lean and Ob/Ob mice were resolved by SDS-PAGE on 5% gels and probed by western blotting using an anti-ApoB antibody reactive with the N-terminus. Arrowheads denote the position of both ApoB100 and ApoB48. C. Total protein abundance from 'B' represented graphically. Statistical significance was calculated using a student t-test (n=3).

Figure. 1 .

 1 Figure. 1. Increased ACF mRNA and protein expression in Ob/Ob liver. A. The abundance of Ob/Ob hepatic apoB, apobec-1 and acf mRNA relative to that quantified from lean control liver was assessed by real time PCR and normalized to β-actin mRNA. Lean animal values were set to 1. Results and standard

Figure. 5 .

 5 Figure. 5. Enhanced ACF abundance supports increases in intracellular ApoB protein. A. Real time PCR was performed on poly A+ purified RNA from cytoplasmic and nuclear subcellular extracts of Ob/Ob and cogenic control animal liver. β-actin mRNA was quantified as a normalization control for RNA input.The relative nuclear and cytoplasmic abundance of apoB mRNA in control mice was set to one. The statistical significance was determined to be p≤0.01 for both cytoplasmic and nuclear differences using a student's t-test with n=3. B. Hepatic cytoplasmic extracts from 3 different lean and Ob/Ob mice were

Fig. 6 .Fig. 7 . 7 A

 677 Fig. 6. Nuclear Retention of ACF Results in Nuclear retention of apoB mRNA and a Reduction of Intracellular and Secreted ApoB Protein. A. Western blot analysis of ACF distribution between the cytoplasm and nucleus following treatment with 5.0 µM cantharidin (10X the IC 50 of PP1) or the solvent, DMSO, used for dissolve the compound. B. The amount of intracellular ApoB protein (ng) per mg of total intracellular protein (open bars) and secreted ApoB protein per mg total protein in the media (closed bars), are shown following treatment of primary rat hepatocytes with cantharidin as in A. ApoB protein was assessed by RIA as previously described. Results and standard deviations are reported where n= 5 (for intracellular) or 8 (for secreted). C. Real time PCR was used to assess the relative abundance of apoB mRNA in the cytoplasm and nucleus, normalized to β-actin, with cantharidin treatment as in parts A & B. The results are representative of 3 trials and statistical significance was calculated using a student ttest.

Primers designed for apoB annealed at nucleotides 3847-3870 and 3893-3917 for left and right primers, respectively, in the 3' end of the transcript. Rat samples assessed by real time PCR used rat Exiqon probe numbers 70 and 18 for apoB

	1, ACCTTCAGCTAACCTTCACACAGT and CGGACTTTGTTTCCTCAGGT; apoB,
	CCAGACAACCTCTTCCTAAAGACT	and	TCAATGTTTATTTTGTTCCTGTTCA;	acf,
	TTTGCCTTTGTGGAATATGAGA		and	GGATGTCCCCACAACTGAAT;	cugbp2,
	CACTGCCCACTTTGTACAGC	and	AGAGGTTTGCCCCCTCTG;	hnrnpa1,
	GCTTTGGCGGTGGTAGTG	and		GCCCAAAATTGGAAGACTGA;	gryrbp,
	GGGGACCAAAGTAGCAGACTC and TGTAGCCTGTTCTTTCCAAAAGT;	and β-actin,
	AAGGCCAACCGTGAAAAGAT and GTGGTACGACCAGAGGCATAC. and actin, respectively, and cDNA was amplified with the following primers: apoB,
	TCAATGTGAAGTATAACGAAGATGG	and	ATGTCCAGATGAGCCTCTCC;	β-actin,
	CCCGCGAGTACAACCTTCT and CGTCATCCATGGCGAACT. Real time reactions were run on
	Applied Biosystems 7900HT Sequence Detection System cycler in 384 well format using cycling
	conditions: 95⁰ C 10 minutes followed by 40 cycles of 95⁰ C for 15 seconds and 60⁰ C for 1 minute, and
	were analyzed using Applied Biosystems SDS2.2 software.

  Galloway et al.ACF Modulates ApoB Expression inHepatocytes 13 deviations were the average of the analysis of 3 livers and statistical significance was calculated using a student's t-test. B. Graphic representation of total hepatic ACF protein abundance normalized to β-actin. Scanning densitometry of western blots probed for ACF and β-actin using ImageJ (NIH) was used to create an ACF to β-actin ratio. For lean animals this ratio was set to one. A representative western blot of ACF and β-actin is shown below the graph. The results are representative of 3 liver preparations and statistical significance was calculated using a student's t-test. Acute leptin treatment suppresses acf mRNA abundance. The relative abundance of acf mRNA in primary mouse hepatocytes, normalized to β-actin mRNA abundance was determined by real time PCR following 4 hours of treatment with increasing concentrations of leptin. Control acf mRNA to β-actin mRNA ratio was set to one. Reduction in acf mRNA to β-actin mRNA ratios at both 1 and 10 ng/mL leptin treatment were statistically significant (p≤0.05 and n=3, where n is the average of one treated 6 mm plate of hepatocytes). Ob/Ob and Db/Db mouse hepatic ACF does not accumulate in the nucleus following insulin administration. Ob/Ob (A) and Db/Db (B) mice were fasted for 12 hours and then injected intraperitoneally with 1.5 IU insulin or saline 4 hours prior to harvesting liver for extract preparation.

	Figure. 2. Figure. 3.

ACCEPTED MANUSCRIPT

Acknowledgements

The authors are grateful to Dr. Alicia Clementi and Allison Gaudy for assistance in the preparation of mouse primary hepatocytes. This work was supported in part by Public Health Services Grants [DK43739, DK78131 and DK38138 awarded to HCS, JDS and RAM].

Supplementary Figure Legends

Table S1. Nuclear to Cytoplasmic ratio (N:C) of ACF and Total ACF in Ob/Ob or C57Bl/6J Mice. Corrected cytoplasmic and nuclear ACF signals were calculated as in Materials and methods by division of the ACF western blot densitometric trace by either β-actin or histone H1densitometric traces, respectively, creating a normalized arbitrary unit (AU). The signal per µg was determined by dividing the AUs by the µg loaded in the lane. Total ACF was determined by multiplying this number by the total µg in the extract.

Figure. S1.

Messenger RNA expression for CUGBP2, GRYBP2 and hnRNPA1 in Ob/Ob liver. Real time PCR was used to quantify the abundance of cugbp2, gryrbp and hnrnpa1 mRNAs in whole liver extracts, relative to β-actin mRNA. The resulting relative abundance and standard deviation were calculated with n=3 and p-values were calculated as in Figure 4. Ob/Ob and lean control values were not significantly different for gryrbp and hnrnpa1. Ob/Ob cugbp2 mRNA was slightly increased above that seen in lean control P>0.03.

Figure. S2. ACF Expression is

Unaltered in Db/Db Mice. A. Real time PCR was used to quantify the abundance of apoB, apobec-1 and acf mRNAs in whole liver extracts, relative to β-actin mRNA. The results are representative of 3 animals and p-values were calculated as in Figure 4. B. Western blot analysis of whole liver cell lysate was used to quantify the abundance of ACF relative to β-actin, as in Figure 4. The resulting relative abundance and standard deviation were calculated with n=3.

Figure. S3. Cytoplasmic ACF Localizes to the Low Density Microsomal Fraction in Rat and in Enhanced

Abundance in Ob/Ob Mouse Liver. A. Western blot analysis of equivalent micrograms of protein from sub-fractionations of cytoplasmic extracts from rat primary hepatocytes, as described [START_REF] Lehmann | Functional characterization of APOBEC-1 complementation factor phosphorylation sites[END_REF]. Blots were probed with a peptide-specific polyclonal antibody recognizing the C-terminus of ACF [START_REF] Sowden | Identification of Novel Alternative Splice Variants of APOBEC-1 Complementation Factor with Different Capacities to Support ApoB mRNA Editing[END_REF], denoted rACF. B. Sub-fractionation of cytoplasmic extracts from Ob/Ob and lean control mice into HDM (Golgi-rich fraction), LDM (ER rich fraction), and residual cytoplasm was probed for ACF using an ACF peptide-specific antibody, denoted mACF. Equivalent micrograms of protein were loaded from each cytoplasmic fraction.


Mathieu Giraud

Stéphane Janot

Jean-Frédéric Berthelot

Charles Deltel

Laetitia Jourdan

Dominique Lavenier

Helene Touzet

Jean-Stéphane Varré

Hélène Touzet

Biomanycores, open-source parallel code for many-core bioinformatics

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Graphics processing units (GPUs) enable efficient parallel processing at a very low cost, and are a first step towards the generalization of massively manycore architectures. Since CUDA (in 2007) and the standard OpenCL (2009), many GPU bioinformatics applications have been developed, from sequence alignment to proteomics or phylogenetics (review in [START_REF] Varré | Advances in Genomic Sequence Analysis and Pattern Discovery[END_REF]).

Biomanycores is a collection of bioinformatics tools, designed to bridge the gap between researches in OpenCL/CUDA high-performance computing on GPU and other "manycore processors" and usual bioinformaticians and biologists.

The main goal is to gather parallel programs and interface them with the BioJava [START_REF] Holland | BioJava: an opensource framework for bioinformatics[END_REF], BioPerl [START_REF] Stajich | The Bioperl toolkit: Perl modules for the life sciences[END_REF] and Biopython [START_REF] Cock | Biopython: freely available Python tools for computational molecular biology and bioinformatics[END_REF] frameworks. We will also provide benchmarks to show in which cases it is worth using parallel versions of the programs.

Biomanycores was presented at BOSC 2009. Since November 2010, a developer is working full-time on this project, redesigning, extending and documenting Biomanycores. The release 1.1104 of Biomanycores (April 15) includes applications for sequence alignment, sequence processing, and RNA folding tools. Next releases are likely to include tools for proteomics, phylogenetics and genome-wide association studies.

Biomanycores design makes it easy to alter existing pipelines and scripts, in order to use a parallel application instead of the standard one. In some cases, this leads to large improvements. For example, the complexity of the brute-force Position Weight Matrix (PWM) scan algorithm is in O(nm), where n is the sequence length and m the matrix size. PWM performance can be measured in millions of operations per second, one operation being the scoring of one sequence character against one matrix column. While the native Biopython search_pwm method peaks at 2 Mop/s, the Biomanycores GPU version (TFM-CUDA) peaks at more than 2 Gop/s, 1000 times faster, even once included the overhead due to the Python interpreter.

In the coming months, we plan to integrate new applications into Biomanycores. Moreover, we wish to receive critical feedback from BioJava, BioPerl and Biopython communities and users in order to improve our interfaces, and discuss further integration into these frameworks.

People who are developing CUDA or OpenCL bioinformatics code (available under a free license) are welcome to get involved in the project -we are willing to help them to integrate their code. Please contact us at contact@biomanycores.org.


B Quertier
email: quertier@obs.u-bordeaux1.fr

S Gauffre

P Caïs

Corrélateurs du Futur Next Generation Correlator

Keywords: ALMA, Corrélateur, Convertisseur Analogique Numérique, Uniboard ALMA, Correlator, Analog to Digital Converter, Uniboard Résumé

Le Laboratoire d'Astrophysique de Bordeaux (LAB) a contribué techniquement au projet ALMA à travers la conception de deux sous-systèmes de l'interféromètre : les convertisseurs analogiques numériques (CAN) et les bancs de filtres numériques. Les spécifications de ces deux sous-systèmes ont une incidence directe sur l'architecture du corrélateur, particulièrement les filtres numériques qui sont à l'origine des nombreux modes d'observation proposés et des performances spectrales de l'instrument. Par ses performances et sa prochaine ouverture à la communauté scientifique, le corrélateur ALMA est un corrélateur du futur, cependant des technologies plus avancées que celles mises en oeuvre pour sa conception sont d'ores et déjà disponibles. De nouveaux développements sont donc en cours au LAB afin de préparer la prochaine génération d'instrument.

Introduction

La question très large « des corrélateurs du futur » sera traitée au travers du prisme des développements techniques réalisés au LAB, en particulier dans le cadre du projet ALMA. Le corrélateur ALMA [1], dont l'ouverture scientifique est programmé pour 2013, propose un niveau de performances et une souplesse d'analyse qui permettent de le considérer comme un parfait exemple d'instrument du futur. L'architecture du corrélateur et les modes d'observations seront décrits. Le sous-système de filtrage numérique appelé Tunable Filter Bank (TFB), développé au sein du LAB, fera l'objet d'une description plus détaillé, car il est au coeur du fonctionnement du corrélateur. Il est aussi le soussystème du corrélateur le plus récent, ce qui permettra une comparaison instructive avec les nouveaux développements techniques réalisés dans le cadre du projet européen Uniboard, financé par le FP7 au travers de Radionet. En effet si les performances du corrélateur ALMA en tant que système sont remarquables et devraient rester inégalées à moyen terme, les technologies mises en oeuvre lors sa conception au cours des années 2000 sont aujourd'hui bien souvent dépassées. Les facteurs d'échelle entre les ressources et les interfaces de la carte ALMA TFB et celles de la carte Uniboard donne une idée du futur des corrélateurs.

L'instrument ALMA

ALMA est un double réseau interférométrique qui combine les signaux millimétriques et sub-millimétriques capturés respectivement par 50 et 16 antennes, déployées sur le site de Chajnantor au Chili. Cet instrument se caractérise donc par une grande sensibilité et de multiples capacités d'imagerie. Les 2 réseaux d'antennes, s'ils disposent de 2 corrélateurs totalement distincts présentent des caractéristiques équivalentes pour toute la partie d'électronique analogique, depuis les récepteurs jusqu'aux CAN. La numérisation du signal s'effectue aux moyens de circuits électroniques spécialement dessinés pour ALMA par le LAB. Il s'agit en effet de circuits fabriqués chez STmicroelectronics permettant de numériser les 264 bandes de base de 2GHz dans 2 polarisations. Cette opération s'effectue à 4 Giga échantillons par seconde (GS/s) et génère des échantillons numériques codés sur 3 bits -8 niveaux. Tous ces signaux numériques sont transmis par fibre optique depuis chacune des 56 antennes jusqu'au bâtiment technique sur le site d'opérations ALMA (AOS) pour être combinés respectivement par le corrélateur du réseau principal et du réseau compact. La suite de l'article se concentre sur le corrélateur du réseau principal auquel le LAB a fortement contribué. Le LAB a développé et produit les 512 cartes Tunable Filter Bank (TFB) permettant aujourd'hui au corrélateur ALMA d'opérer dans les deux modes d'analyse TDM et FDM pour un réseau maximum de 64 antennes. La Figure 1 ci-après représente les principales fonctions du système ALMA depuis la numérisation du signal et principalement au sein du corrélateur. Chaque bande de base de 2 GHz est numérisée, transmise par fibre optique, 32 fenêtres d'analyse indépendantes de 62.5MHz peuvent ensuite être sélectionnées grâce au système TFB avant d'être individuellement corrélée (en mode FDM). Un processus mathématique complexe permet ensuite (software post-corrélation) de recoller les différentes sous-bandes afin de représenter avec une grande précision la fenêtre effective souhaitée par l'astronome.

Le corrélateur ALMA

Détails techniques

Le transport du signal au sein du corrélateur est particulièrement problématique. Le système complet doit corréler toutes les lignes de base d'un réseau comportant un maximum de 64 antennes générant 4 bande de base x 2 polarisations x 32 sous-bandes. La transmission des signaux entre cartes s'effectue en différentiel à 250MHz, et nécessite 16384 câbles. La phase du signal en sortie de chaque câble est vérifiée et ajustée automatiquement afin d'obtenir une transmission optimale. Le corrélateur est organisé en quadrants, chaque quadrant traite le signal capturé par l'ensemble du réseau d'antenne pour une bande de base et 2 polarisations. Les modes « basiques » présentés précédemment s'appliquent à des fenêtres uniques de dimension comprise entre 31.25MHz et 2GHz. Le corrélateur ALMA offrira également à terme (certains développements du système informatique sont encore nécessaires) des modes plus évolués, notamment :

-le corrélateur peut opérer des analyses simultanées sur plusieurs fenêtres disjointes, le nombre de fenêtres étant limité à 4 et chaque fenêtre doit avoir une dimension multiple de 62.5MHz. -des modes multi-résolutions spectrales sont également possibles, chaque quadrant du corrélateur peut en effet être découpé en plusieurs sous-unités opérant dans des modes basiques différents. -Des sous-réseaux d'antennes pourront également être définis et les ressources du corrélateur seront partagées pour former des corrélateurs indépendants opérant sur chaque sous-réseau. Certains modes « avancés » envisagés ne sont pas encore pleinement supportés par le système informatique et la faisabilité de certains doit encore être étudiée, notamment pour ce qui concerne les débits de données générés. A ce titre le corrélateur ALMA demeure un instrument du futur.

La future génération d'instrument

Les enjeux

Bien qu'à l'état de l'art au moment de leur conception, de nombreux sous-systèmes de l'instrument ALMA utilisent des technologies, qui ne sont plus aujourd'hui, les plus performantes. En effet, la plupart des cartes électroniques du corrélateur ont été développées il y a environ 10 ans, la plus récente (la carte TFB) a été produite entre 2006 et 2008. Les ASICs ALMA (CAN et corrélateur) utilisent une technologie 0.25µm, les FPGAs du corrélateur utilisent le plus souvent des technologies de l'ordre de 0.15µm, le système TFB une technologie 90nm. Aujourd'hui des technologies CMOS 65nm sont disponibles pour développer des ASICs et les FPGAs les plus récents sont fabriqués en 28nm. Une nouvelle génération de CAN permettrait de numériser des bandes encore plus larges, donc de simplifier grandement le dessin des systèmes de découpe et descente en fréquence, mais aussi d'augmenter la sensibilité du corrélateur en travaillant avec une plus grande dynamique de signal. En particulier un CAN capable de numériser la totalité de la bande de 8GHz délivrée par les récepteurs permettrait de s'affranchir totalement des étages de découpe et descente en fréquence du back-end, améliorant la reproductibilité des traitements opérés sur les différents bras de l'interféromètre. De nouveaux prototypes de CAN sont actuellement en cours de développement au LAB, les fréquences visées sont au minimum de 8GS/s et le format supérieur à 3 bits. Les finesses de gravure des composants numériques ont également largement évoluées, modifiant les possibilités d'analyse spectrale des prochains corrélateurs. Enfin la transmission des signaux numériques a été révolutionnée par la démocratisation des liens série rapides, or les débits de données générés par un radiotélescope (notamment dans le corrélateur) constituent un défi pour chaque génération d'instrument. A l'époque l'architecture XF avait été préférée et sélectionnée par l'équipe du NRAO, notamment sur des critères liés au nombre de connexions physiques à réaliser pour transporter le signal au sein du corrélateur.

L'exemple du projet Uniboard

Dans le cadre du projet Radionet / FP7, un consortium d'instituts * développe depuis 2009 une carte numérique générique pour le traitement du signal en radioastronomie. Cette carte, de taille standard utilise des composants numériques programmables (FPGAs) à l'état de l'art afin d'offrir une très grande puissance de calcul et des débits d'interface très élevés. Des algorithmes de traitement du signal (firmwares) sont également développés dans l'optique de rénover le corrélateur de JIVE. Cette plateforme devrait également être utilisée pour les projets APERTIF et LOFAR. Les principales fonctionnalités envisagées sont : « digital baseband converter », « beamformer », « RFI mitigation », « correlation ». La Figure 3 donne un aperçu de la carte Uniboard, peuplée de 8 FPGAs, de modules de mémoire externe et disposant de diverses interfaces. La consommation grossièrement estimée à ce jour pour la carte est de 280W.

Figure 4. Représentation schématique de la carte Uniboard

La Table 3 ci-après donne un aperçu des ressources disponibles respectivement avec la carte ALMA TFB et avec la carte Uniboard, elle fait apparaître en moyenne un facteur 10 entre les deux. Si on écarte de la comparaison les modules de mémoire externe (dont la carte TFB n'est pas équipée), le facteur d'échelle le plus important concerne les débits potentiels des interfaces. En effet entre les 2 générations de FPGAs, la principale différence concerne l'apparition de liens série rapides, les autres types de ressources ayant « simplement » bénéficiés de l'avancée de la technologie. Ces nouveaux liens rapides supportent de nombreux protocoles de communication et leur utilisation devrait impacter grandement la transmission des signaux numériques des CANs vers les corrélateurs et en interne aux corrélateurs, de carte à carte, ou entre composants numériques. Le LAB qui poursuit ses développements liés aux CANs et aux systèmes de traitement numérique, prête une attention particulière à cette nouvelle technologie. Dans le cadre d'Uniboard, le LAB a dessiné en collaboration avec Arcetri un nouveau système de filtrage [START_REF] Comoretto | Uniboard digital receiver initial design[END_REF], dont les performances sont sensiblement 2 fois supérieures à celles d'ALMA. (une version avec un gain de facteur 4 est prévue). Précisons que ce « design » n'utilise qu'une faible proportion des ressources disponibles : pas de mémoire externe et moins de 50% des ressources FPGAs. Enfin les micro-contrôleurs embarqués sont apparus dans les dernières années de développement d'ALMA, ils sont maintenant des fonctions usuelles dans les FPGAs. Leur utilisation devrait permettre de simplifier la problématique de contrôle et de paramétrage des grands corrélateurs. En effet la disponibilité d'un grand nombre de modes d'observations est synonyme de très nombreux paramètres à configurer dans l'ensemble des composants numériques opérant au sein du système de filtrage et du corrélateur.

Carte

L

  'architecture initialement imaginée par le NRAO correspond à un corrélateur de type XF (corrélation puis transformée de Fourier) où les fonctions de corrélations sont calculées sur la largeur totale de la bande numérisée. L'électronique fonctionnant à 125MHz alors que le signal doit être traité à 4GS/s, 32 plans de corrélateur travaillant en parallèle sur des tranches temporelles adjacentes de signal sont nécessaires pour réaliser l'analyse spectrale. Ce mode d'analyse est appelé Time Division Mode (TDM). Dans le concept européen, initialement proposé dans le contexte d'un corrélateur de seconde génération, la bande numérisée est découpée en tranches fréquentielles (sous-bandes), individuellement corrélées, l'architecture est dite HXF (pour Hybrid XF). Ce mode d'analyse appelé Frequency Division Mode permet d'adapter la vitesse du corrélateur à la largeur de bande à traiter et ainsi d'offrir des résolutions spectrales extrêmement fines sur de très larges bandes (de quelques kHz à quelques MHz sur des bandes de quelques dizaine de MHz à plusieurs GHz) et une grande souplesse d'analyse[START_REF] Quertier | Enhancing the Baseline ALMA Correlator Performances with the Second Generation Correlator Digital Filter System[END_REF]. La complexité du système de filtrage numérique traitant le signal en amont du corrélateur est la contrepartie à de telles performances.

Figure 1 .

 1 Figure 1. Schéma fonctionnel du corrélateur ALMA (adapté de Escoffier R., Webber J. and Baudry A. in ALMA-60.00.00.00-001-C-SPE document)

Figure 2 .

 2 Figure 2. Photographie (tiré de [1]) d'un quadrant du corrélateur dans le bâtiment technique de l'AOS sur le site ALMA. Le système complet comprend 4 quadrants identiques.Un autre point critique est la dissipation de puissance qui impacte la fiabilité à long terme du corrélateur. Cette question a fait l'objet de nombreux efforts afin d'optimiser la circulation d'air au sein du corrélateur mais aussi en terme d'algorithme implanté dans les composants électroniques. Au final le système corrélateur complet dissipe 130kW pour une puissance de calcul de 17 x 10 15 opérations par seconde pour la corrélation 2 bits et environ 10 15 multi-bits opérations par seconde pour le système de filtrage. Le système TFB a été particulièrement concerné par cette problématique car s'il a permis d'accroitre les performances du concept initial d'un facteur 32, il nécessite également une puissance de calcul importante. Dans sa première version une carte TFB dissipait 150W, un changement de technologie et un nouvel algorithme ont permis de limiter la consommation à 60W et de conserver la température de jonctions des composants FPGAs dans la gamme conseillée (<70°C)[START_REF] Camino | The new 3 stage, low dissipation digital filter of the ALMA correlator[END_REF]. Le traitement du signal implanté dans les FPGAs pour extraire une sous-bande est représenté schématiquement en Figure3: le signal est d'abord traité par un système oscillateur local / mélangeur permettant un positionnement spectral de la fenêtre d'analyse, le signal complexe généré est traité identiquement sur les voies réelles et imaginaires par 3 étages de filtrages cascadés, le signal est ensuite converti en signal réel et re-quantifié pour s'adapter au format de corrélation.

2.1. Architecture du corrélateur ALMA Les

  principales spécifications du corrélateur ALMA sont résumées dans la table 1 ci-après. Le corrélateur permet l'analyse d'une très large bande (16GHz à travers deux polarisations) pour 64 antennes et offre de nombreuses possibilités spectrales : résolution fine ou large, positionnement libre des fenêtres d'analyse et diverses sensibilités.

		Spécification
	Antennes	≤ 64
	Bande de base par antenne	8 x 2GHz
	Numérisation	3 bits / 8 niveaux à 4G S/s
	Format de corrélation	2 bits / 4 niveaux, 4 bits / 16 niveaux, 3 bits / 8 niveaux
	Horloge	125MHz
	Ligne de base maximale	30km
	Nombre de points spectraux par bande de base (FDM)	≤ 8192
	Nombre de points spectraux par bande de base (TDM)	≤ 256
	Produits de polarisation	1, 2 ou 4

Table 1 .

 1 Principales spécifications du corrélateur ALMA

Table 2 .

 2 Le corrélateur ALMA supporte deux modes d'observations principaux : TDM et FDM. Les modes TDM sont idéaux pour des observations nécessitant des « dump time » rapides (16ms en cross-corrélation) et des résolutions spectrales « modestes ». Les nombreux modes FDM permettent d'obtenir de très bonnes résolutions spectrales nécessaires aux observations spectroscopiques sur diverses largeurs de bande. Il existe également plusieurs possibilités en termes de sensibilité : en FDM des modes « twice nyquist » et « 4 bits x 4 bits » permettent d'accroitre la sensibilité au détriment de la résolution, de la largeur de bande ou du nombre de produits de polarisation, comme les modes « 3 bits x 3 bits » en TDM. D'une manière générale les 71 modes proposés par le corrélateur correspondent à une répartition différente des ressources du corrélateur sur les sous-bandes générées par le système de filtrage. Initialement, seuls 21 modes seront disponibles pour les premières observations scientifiques «ALMA Early Science » (cf Table2). Les 21 modes d'observation disponibles pour ALMA Early Science.Pour une largeur de bande donnée la résolution spectrale dépend du nombre de produits de polarisation (1, 2 ou 4).

	Digital LO
	and Mixer
	x
	DDS
	LO

Table 3 .

 3 Comparatif des cartes ALMA TFB et Uniboard4. ConclusionEntre 2008 et 2010, les 3 premiers quadrants du corrélateur ALMA ont été installés dans le bâtiment technique de l'AOS. Pour l'ouverture à la communauté scientifique, prévue en 2013, des opérations à 2 quadrants seront possibles. Elles permettront, associées à un système de contrôle software adéquat actuellement en développement, de traiter jusqu'à 32 antennes et 4 bandes de base. A terme, le corrélateur ALMA offrira 71 modes d'observations « basiques » et une multitude de modes avancés dont le multifenêtrage, la multi-résolution et le « sub-arraying ». Ces fonctionnalités avancées et les performances spectrales du corrélateur ALMA sont remarquables et font de ce système un instrument du futur. Cependant de nouvelles technologies sont d'ores et déjà disponibles et permettent d'envisager une nouvelle génération d'instruments. Évidemment l'accroissement de la puissance de calcul, liée à la finesse de gravure, est un enjeu essentiel, mais l'apparition des liens série rapides pourrait s'avérer plus importante encore, car elle permet de reconsidérer l'architecture même du corrélateur. Les microcontrôleurs embarqués, quant à eux, devraient faciliter les opérations de contrôle et de paramétrage de ces instruments toujours pus versatiles.

		ALMA TFB	Carte Uniboard	Facteur d'échelle
	Nombre de FPGAs	16	8	0.5
	Technologie	90nm	40nm	~0.5
	Cellules logiques	~35k x 16	~180k x 8	~2.5
	Mémoires	~1.4Mb x 16	~30Mb x 8 (+32Gb x 16 * )	~10
	MAC	32 x 16	644 x 8	~10
	Interface	160 lignes ~125MHz	128 lignes ≤ 1.6Gb/s	~40
			64 lignes ≤ 6.375Gb/s
			16 lignes ≤ 10.325Gb/s