{
    "v1_Abstract": "1",
    "v1_col_introduction": "introduction  : RNA editing is a post-transcriptional regulatory mechanism which often results in conversion of adenosine to inosine (A-to-I) in mRNA sequences (Nishikura 2010). Recent whole transcriptome sequence analyses reveal abundant A-to-I type RNA editing in non-coding and Alu-containing transcript regions while RNA editing of protein encoding regions, especially of non-A-to-I types, appears to be very rare (Kleinman, Adoue & Majewski 2012; Piskol et al., 2013). Site-specific C-to-\n38\n39\n40\n41\n42\n43\n44\n45\n46\n47\n48\n49\n50\n51\n52\n53\nPeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013)\nR ev ie w in g M an\nus cr ip t\nU RNA editing leading to amino acid recoding of an endogenous gene in normal mammalian cells was previously confirmed, to our knowledge, only in the ApoB gene encoding apolipoprotein B (Blanc & Davidson 2003). C-to-U editing of Apo B is catalyzed by APOBEC1 cytidine deaminase that generates a shorter protein isoform in intestinal epithelial cells. APOBEC1 also inactivates 4- 17% of neurofibromatosis type 1 (NF1) gene transcripts by site-specific C-to-U RNA editing in certain high-grade NF1 tumors but not in normal cells (Skuse et al., 1996).\nWe previously identified SDHB C-to-U mRNA editing (C136U) at low steady-state levels in peripheral blood mononuclear cells (PBMCs) of normal individuals using reverse transcription (RT) and semi-quantitative PCR (Baysal 2007). Analysis of the purified PBMC subsets obtained from a single donor showed that the C136U editing rate was higher in monocytes than in lymphocytes. SDH is a central metabolic enzyme in Krebs cycle (Rutter, Winge & Schiffman 2010) and a tumor suppressor for hereditary paraganglioma (PGL) (Baysal et al., 2000). SDH catalyzes the oxidation of succinate to fumarate during aerobic respiration. In anaerobically respiring mitochondria of lower organisms, SDH is inactive and fumarate is often reduced to succinate in a reverse reaction catalyzed by fumarate reductase (Muller et al., 2012). Germline inactivating mutations in the nuclear-encoded SDH subunit genes, primarily in SDHB and SDHD, cause PGL tumors (Burnichon et al., 2012) which show constitutive activation of the hypoxia-inducible pathways. PGL tumors recapitulate the high-altitude associated carotid body (CB) paragangliomas (Saldana, Salem & Travezan 1973), show increased severity with increased altitudes (Astrom et al., 2003; Cerecer-Gil et al., 2010) and share transcriptome characteristics with Von-Hippel Lindau (VHL) disease tumors (Dahia et al., 2005; Lopez-Jimenez et al., 2010). The VHL gene product plays an important role in normoxic degradation of hypoxia-inducible factors (HIFs) (Kaelin & Ratcliffe 2008). Succinate and reactive oxygen species that accumulate upon SDH inactivation were implicated as downstream messengers leading to stabilization of HIF1\u03b1 in normoxic conditions (Selak et al., 2005; Guzy et al., 2008). However,\n54\n55\n56\n57\n58\n59\n60\n61\n62\n63\n64\n65\n66\n67\n68\n69\n70\n71\n72\n73\n74\n75\n76\n77\nPeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013)\nR ev ie w in g M an\nus cr ip t\nmechanisms of PGL tumorigenesis remains unconfirmed partly because animal or cell culture models that link SDH mutations and hypoxia are lacking.\nThe SDHB C136U RNA editing converts a highly conserved arginine residue to a premature stop codon (R46X) shortly after the mitochondrial targeting signal and inactivates the SDHB gene product, the iron-sulfur subunit of the SDH complex. The R46X germ line mutation was previously described in multiple index patients with PGL confirming its pathogenicity (Bayley, Devilee & Taschner 2005). Here, we studied the SDHB C136U transcript editing in short-term cultured monocyte-enriched PBMCs using a novel allele specific quantitative PCR assay (AS qPCR), highthroughput genetic methods, immunophenotyping, immunoseparation, morphology and database analysis. Our aim was to determine the prevalence, distribution, cell type origin and other factors influencing SDHB editing using a reproducible assay to gain functional insights. We found that SDHB C136U editing is present at low levels in fresh uncultured total and monocyte-enriched PBMCs, but it markedly increases during normoxic macrophage differentiation in vitro and upon short-term hypoxic exposure in monocytes. These results provide unprecedented evidence that functional transcript dosage for a central metabolic enzyme is regulated by environmentallyinducible site-specific C-to-U RNA editing.",
    "v2_col_introduction": "introduction  : RNA editing is a post-transcriptional regulatory mechanism which often results in conversion of adenosine to inosine (A-to-I) (Nishikura 2010). Recent whole transcriptome sequence analyses reveal abundant A-to-I type RNA editing in non-coding and Alu-containing transcript regions while RNA editing of protein encoding regions, especially of non-A-to-I types, appears to be very rare (Kleinman, Adoue & Majewski 2012; Piskol, et al., 2013). Site-specific C-to-U RNA editing leading to amino acid recoding of an endogenous gene in normal mammalian cells was previously confirmed, to our knowledge, only in the APOB gene encoding apolipoprotein B (Blanc & Davidson 2003). C-to-U editing of APOB is catalyzed by APOBEC1 cytidine deaminase that generates a shorter protein isoform in intestinal epithelial cells. APOBEC1 also inactivates 4%-17% of neurofibromatosis type 1 (NF1) gene transcripts by site-specific C-to-U RNA editing in certain high-grade NF1 tumors but not in normal cells (Skuse, et al., 1996).\nWe previously identified SDHB C-to-U mRNA editing (C136U) at low steady-state levels in peripheral blood mononuclear cells (PBMCs) of normal individuals using reverse transcription (RT) and semi-quantitative PCR (Baysal 2007). Analysis of the purified PBMC subsets in a single donor showed that the C136U editing rate was higher in monocytes than in lymphocytes. SDH is a central metabolic enzyme in Krebs cycle (Rutter, Winge & Schiffman 2010) and a tumor suppressor for hereditary paraganglioma (PGL) (Baysal, et al., 2000). SDH catalyzes the oxidation of succinate to fumarate during aerobic respiration. In anaerobically respiring mitochondria of lower organisms, SDH is inactive and fumarate is often reduced to succinate in a reverse reaction catalyzed by fumarate reductase (Muller, et al., 2012). Germline inactivating mutations in the nuclear-encoded SDH subunit genes, primarily in SDHB and SDHD, cause PGL tumors (Burnichon, et al., 2012) which show constitutive activation of the hypoxia-inducible pathways. PGL tumors recapitulate the\n2\n16\n17\n18\n19\n20\n21\n22\n23\n24\n25\n26\n27\n28\n29\n30\n31\n32\n33\n34\n35\n36\n37\n38\nPeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013)\nR ev ie w in g M an\nus cr ip t\nhigh-altitude associated carotid body (CB) paragangliomas (Saldana, Salem & Travezan 1973), increase in severity with increased altitudes (Astrom, et al., 2003; Cerecer-Gil, et al., 2010) and share transcriptome characteristics with Von-Hippel Lindau (VHL) disease tumors (Dahia, et al., 2005). The VHL gene product plays an important role in normoxic degradation of hypoxia-inducible factors (HIFs) (Kaelin & Ratcliffe 2008). Succinate and reactive oxygen species that accumulate upon SDH inactivation were implicated as downstream messengers leading to stabilization of HIF1\u03b1 in normoxic conditions (Selak, et al., 2005; Guzy, et al., 2008) although mechanisms of PGL tumorigenesis remains unconfirmed.\nThe SDHB C136U RNA editing converts a highly conserved arginine residue to a premature stop codon (R46X) shortly after the mitochondrial targeting signal and inactivates the SDHB gene product, the iron-sulfur subunit of the SDH complex. The R46X germ line mutation was previously described in multiple index patients with PGL confirming its pathogenicity (Bayley, Devilee & Taschner 2005). Here, we studied the SDHB C136U transcript editing in short-term cultured PBMCs using a novel allele specific quantitative PCR assay (AS qPCR), high-throughput genetic methods, immunophenotyping, immunoseparation, morphology and database analysis. Our aim was to determine the prevalence, distribution, cell type origin and other factors influencing SDHB editing using a reproducible assay to gain functional insights. We found that SDHB C136U editing is present at low levels in fresh uncultured total and monocyte-enriched PBMCs, but it markedly increases during macrophage differentiation in vitro and upon short-term hypoxic exposure in monocytes. These results provide unprecedented evidence that functional transcript dosage for a central metabolic enzyme is regulated by environmentally-inducible site-specific C-to-U RNA editing.\n3\n39\n40\n41\n42\n43\n44\n45\n46\n47\n48\n49\n50\n51\n52\n53\n54\n55\n56\n57\n58\n59\nPeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013)\nR ev ie w in g M an\nus cr ip t",
    "v1_text": "materials and methods : Leukocyte isolation and cell culture Leukocytes were isolated from Trima leukoreduction filters (Terumo BCT, Lakewood, CO) of anonymous healthy platelet donors following an IRB-approved protocol. PBMCs were purified by histopaque 1077 (Sigma-Aldrich, St. Louis, MO) and washed twice with RPMI-1640/10% fetal bovine serum (FBS) to remove residual platelets. Each filter frequently gave 5X108 or more PBMCs. Monocytes were enriched by the cold aggregation method (Mentzer et al., 1986) with certain 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t modifications. PBMCs were suspended in 35 ml RPMI-1640 in a 50 ml polypropylene tube and incubated at 4 oC for 1 h on a rocker panel for homotypic aggregation of monocytes. For monocyte enrichment, the tube was positioned upright, underlaid with 6 ml FBS and incubated overnight at 4 oC. The cell precipitate under the serum was collected for culture. Monocytes enrichment was approximately 70% (n=5) by short-term precipitation (up to 3 hours) and 27% (n=10) by overnight precipitation over serum as determined by percentages of the CD14+ cells by flow cytometry. The non-aggregating upper layer was markedly depleted of monocytes (less than 4%, n=3). We used the overnight precipitation method which gave higher yield of total cells and led to more consistent development of adherent aggregates that were associated with higher levels of C136U editing in culture. Precipitated cells under the serum were centrifuged for 10 min at 250xg and suspended in 20 ml RPMI-1640 with 10% FBS and penicillin/streptomycin. Cell density was calculated by a hemocytometer (Neubauer improved, InCyto, Covington, GA). After further dilution, two milliliters of the cell suspension was distributed to each well of a six-well tissue culture plate (Costar, Corning Incorporated, Corning, NY). Neutrophils were isolated as described (Maqbool et al., 2011) from the precipitated cell fraction containing red cells after histopaque 1077 centrifugation of the leukoreduction filter cells. Giemsa staining showed that approximately 80% of the isolated cells were granulocytes and about 20% were lymphocytes. Editing rates were determined in daily collected individual wells. We cultured 21-30x106 cells/2 ml per well (also see results). Cultures were incubated at, 5% CO2 with either 21% O2 (normoxia) or 1% O2 combined with 94% nitrogen (hypoxia). Hypoxic cultures were pre-incubated at 37 oC, 21% O2 for 2-3 hours before placing them in the hypoxia chamber (XVIVO system, BioSpherix). Fresh culture media was added after 7 days in culture. Lymphoblastoid cell line RNAs were isolated from previously described EBV-transformed PBMCs (Baysal 2007,Baysal et al., 2000). HEK 293T embryonic kidney cell line and THP-1 monocytic 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t leukemia cell line was purchased from ATCC (Manassas, VA). Various cytokines/differentiating agents were added for the following working concentrations: m-CSF (50 ng/ml), GM-CSF (50 ng/ml), IL4 (50 ng/ml, vitamin D (10 nM), retinoic acid (1 \u00b5M), PMA (4 beta-phorbol 12-myristate 13-acetate) (100 nM). Cytokines were purchased from Peprotech (Rocky Hill, NJ) and differentiating agents were purchased from Sigma-Aldrich. Lipopolysaccharide (List Biological Libraries Inc, Campbell, CA) is used at a final concentration of 100ng/ml. results and discussion : Analysis of C136U mutation rate in PBMCs by RT and AS qPCR To determine the prevalence, distribution, cell type origin and other factors influencing SDHB editing, we developed a highly specific and reproducible assay for allele specific quantitative PCR amplification (AS qPCR) of the total and edited C136U SDHB mRNAs (see methods). Using this assay, we found that the C136U transcripts were very low or absent in B cell lymphoblastoid and embryonic kidney cell lines. The editing occurred at slightly higher levels in PBMCs isolated from fresh blood than in the cell lines (Figure 1A). To study the relative contribution of monocytes and lymphocytes to SDHB editing, we performed monocyte enrichment using the well-established cold aggregation method (Mentzer et al., 1986) with certain modifications (see methods). This method essentially separates PBMCs into cold-aggregating monocyte-enriched and non-aggregating monocyte-depleted (i.e., predominantly lymphocytic) compartments. Monocyte-enriched samples by the cold aggregation method showed higher editing rates than the matched PBMCs (2.16% versus 1.48%, n=36, p=1.6X10-5, Wilcoxon matched pairs signed ranks test) and than the matched monocyte-depleted samples (1.47% versus 0.58%, n=10, p=0.005, Wilcoxon matched pairs signed ranks test) (Figure 1A). To further confirm monocyte-origin of the edited transcripts, we isolated peripheral blood monocytes to >92% purity by CD14+ microbeads and tested the mutation rates in three samples. In each sample, a higher mutation rate was found in the monocytes than the CD14lymphocytes (average 0.84% vs 0.38%). A positive but weak correlation was found between the CD14+ monocyte-fraction and the C136U editing rate in cold-aggregated PBMC samples (Figure 1B). A linear regression model provided a point estimate of 4.25% C136U editing rate in a pure monocytic cold-aggregated population. These results indicate that C136U editing occurs in freshly isolated monocytes in low but statistically significantly higher rates than in lymphocytes. 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t figure legends : Figure 1 SDHB C136U RNA editing is induced in monocytes during short-term culture. (A) C136U editing is measured in 9 lymphoblastoid cell lines and one embryonic kidney cell line, freshly isolated PBMCs (n=50), PBMCs monocyte-enriched by cold-aggregation (Mono+; n=42) and monocyte-depleted PBMCs comprised of largely lymphocytes (Mono-; n=10). Horizontal lines represent mean \u00b1 standard errors throughout the figures. (B) Fraction of the C136U transcripts weakly correlates with the CD14+ monocyte percentage in monocyte-enriched PBMCs (Pearson correlation coefficient r=0.36). (C) Impact of short-term culture on C136U editing. Short-term culture of five monocyte-enriched PBMCs shows upregulation of C136U editing in adherent cells in culture days 5-7 compared to days 1-3. Day 0 represents the uncultured samples. (D) Flow cytometric sorting of CD14+ versus CD14- adherent cells on culture days 5-8 shows higher mutation rates in monocyte-macrophage lineage than in lymphocytes (p=0.04, n=5, Wilcoxon matched pairs signed ranks test). (E) C136U editing is lower among non-adherent cells in the supernatant than in adherent cells (4.3% versus 9.8%) on culture days 5-7 (n=19 wells from 11 samples on days 5-7, p=2.1 X10-4, Wilcoxon matched pairs signed ranks test). Figure 2 SDHB C136U RNA editing correlates with adherent monocyte-rich adherent aggregates (AA) in culture. The AAs on culture days 1 (A) and 5 (B) are shown (5X low power field; vertical bars equal to 1 mm). The culture is initiated with 30 million cells per well. The AAs grow in size until approximately days 5-7 when the C136U editing rate also usually peaks. (C) High editing rates are seen in cultures with intermediate and high density of adherent aggregates. Aggregate densities are grouped as follows: Low= less than 10 aggregates/per 5X low power field (lpf); Intermediate=1020 aggregates/lpf; High= more than 20 aggregates/lpf (D) Giemsa stain highlights an adherent aggregate on day 6 culture. Individually attached mature macrophages have mostly round eccentric 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t nuclei and abundant cytoplasm. Occasional multi-nucleated macrophages (arrowheads) and rare small lymphocytes (green arrows) are also present. Macrophages within the aggregate are smaller with less cytoplasm and occasionally have curved nuclei (arrows). (E) CD163 immunostaining of adherent aggregates. Immunocytochemical staining for monocyte-macrophage-specific antigen CD163, a scavenger receptor for hemoglobin-haptoglobin complex, demonstrates that the adherent aggregates are primarily comprised of monocyte/macrophage lineage cells. Individually attached macrophages outside the aggregate also stain with variable intensity (short arrows); while numerous small lymphocytes are negative (long arrows). Figure 3 SDHB C136U RNA editing increases during monocyte-macrophage maturation. Flow cytometric evaluation of monocyte-macrophage maturation is shown in uncultured cold aggregated PBMCs (first column), adherent cells on culture day 6 (second column) and adherent cells on culture day 15 (third column). The day 15 culture shows a relatively homogenous large (indicated by high forward scatter, FSC-A) macrophage population that has high complexity (indicated by high side scatter, SSC-A) and is positive for CD206 (mannose receptor), HLA-DR and CD163. In contrast, uncultured monocytes are largely a uniform population that is smaller with less complexity and is negative for CD206. The day 6 culture, which has 11.6% C136U editing rate, shows a CD14+ population that is brightly positive for HLA-DR, dim-positive for CD206 and negative for CD163. CD14+ monocyte-macrophage and lymphocyte populations are marked by green and red, respectively. Figure 4 Hypoxia induces SDHB C136U RNA editing. (A) The editing rates are higher on days 1-3 in hypoxia than the average of adherent and supernatant cells in normoxic cultures (3-day average 11.2% in hypoxia versus 3.6% in normoxia; p=0.006, n=4, Wilcoxon matched pairs signed ranks test). In contrast, as the hypoxic editing rate decreases along with extensive cell 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t death on days 5-7, it increases in normoxic cultures as previously described (Figure 1C). The average of four independent cultures is presented. (B) C136U editing rates in 14 independent cultures in hypoxia versus normoxia during 3 days of culture are shown. The editing rates were obtained from supernatant in all hypoxic cultures (first column) and from total cell population in normoxic cultures, except in two normoxic cultures where only supernatant was collected (second column). D0 samples represent the original uncultured cold aggregated PBMCs. Hypoxia statistically significantly increased the C136U editing rates in days 1, 2 and 3. (C) PCR amplification and Taq1 restriction enzyme (RE) digestion shows no evidence of C136T DNA mutation in SDHB exon 2 genomic DNA (gDNA) (right panel) in the same samples with high C136U RNA editing rates (left panel). The C136U RNA editing rates above the lanes were measured by AS qPCR. RT- and gDNA-PCRs generate amplicon sizes of 285 bp and 233 bp, respectively. Taq1 RE digestion of wild type cDNA and gDNA sequences generates 159 bp/126 bp and 131 bp/102 bp bands, respectively. C136U/T mutation destroys the RE site. (D) Western blot analysis of normoxic (N) and hypoxic (H) cultures from one donor shows no major changes in SDHB protein expression, normalized against beta actin, as the editing rate increases in hypoxia, but possibly a subtle decrease when the C136U percentage is 16.1% on day 2 in hypoxia. (E) The editing rates are higher in flow sorted CD14+ viable cells than CD14- viable cells from day 2 hypoxic cultures (average 9.35% versus 1.63%; p<0.04, n=4, Wilcoxon matched pairs signed ranks test, one-sided). In all experiments, hypoxic and control normoxic cultures derive from the same donors (i.e., oxygen tension is the only variable). 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t statistical analysis : Unless specified otherwise, statistical analyses of experimental data was performed using the online interface of SISA at http://www.quantitativeskills.com/sisae (Uitenbroek) using two-tailed nonparametric tests. Graphics and descriptive statistics were generated in Excel (version 10; Microsoft, Redmond, WA). Means and associated standard errors are depicted by horizontal bars in the figures. 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t 2 day 3 1.3% nr : 3 day 3 4% nr : conclusions : We found that an acquired RNA nonsense mutation (R46X) introduced by C-to-U RNA editing dynamically reduces the SDHB functional transcript dosage in monocytes during early macrophage differentiation in normoxia and upon short-term exposure to hypoxia. The fraction of C136U transcripts was variable in monocyte-enriched PBMC cultures and peaked around 19% in normoxic cultures but increased to up to 49% within 2 days in hypoxia. The editing rates must be higher in monocytes because CD14+ cell isolation increased the percentages of C136U transcripts by 1.25-fold in normoxic cultures and 1.68-fold in hypoxic cultures. In contrast, no other cell type showed convincing evidence of SDHB RNA editing. Cytokine-driven differentiation of monocytes to 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t macrophages or dendritic cells, or LPS stimulation of monocytes reduced the C136U editing rates under normoxia. Gene expression analyses identified CDA as a candidate gene for SDHB editing. To our knowledge, these results provide the first examples of hypoxia-inducible coding RNA editing and a programmed mutation targeting an endogenous nuclear gene in myeloid cells. On the basis of genetic studies on SDH-mutated paragangliomas that show constitutive activation of hypoxia-inducible pathways, we hypothesize that reduction in the SDHB dosage by RNA editing facilitates hypoxia adaptation in monocytes by amplifying hypoxia signaling from mitochondria. The programmed SDHB RNA editing during normoxic macrophage differentiation may serve to augment signaling of inflammatory hypoxia. Some parallels can be drawn between the monocytes circulating in the high-oxygen environment of the peripheral blood and subsequently migrating into the hypoxic areas where differentiation into macrophages occurs versus certain facultatively aerobic organisms such as the intestinal parasitic worm Ascaris Suum (A. Suum). A. suum spores living in open air are exposed to high oxygen concentrations and utilize SDH; whereas parasitic adult worms living in the hypoxic environment of intestine use fumarate reductase (Kita et al., 2002). Thus, suppression of SDH might contribute to increased monocyte/macrophage survival in hypoxia. Because SDHB protein levels do not appear be altered significantly, it is conceivable that other hypoxia-inducible changes may also contribute to enhanced glycolysis observed in hypoxic monocytes (Roiniotis et al., 2009). The mechanisms linking hypoxia sensing and signaling to SDHB RNA editing remain to be determined. It is conceivable that HIF transcriptional activity is required for induction of one or more components of putative cytidine deaminating RNA editor. Whether hypoxia-sensing leading to SDHB RNA editing in monocytes originates in the oxygen-dependent prolyl hydroxylase enzymes that post-translationally regulate hypoxic stabilization of HIFs also remains to be confirmed (Kaelin & Ratcliffe 2008). The method described here will allow exploration of these mechanistic questions. 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t More broadly, our results extend previous observations associating external factors with differential A-to-I RNA editing (Garrett & Rosenthal 2012; Sanjana et al., 2012; Balik et al., 2013) and demonstrate in a robust experimental model that coding RNA editing of certain genes occurs dynamically in a cell type and environment-dependent manner. Our findings suggest that gene targets of RNA editing can be missed by whole transcriptome sequence analyses unless tissues are examined in their physiologically relevant states. If the threshold for significant RNA editing is set at 10% or more in whole transcriptome sequence analyses (for example, (Park et al., 2012)), the SDHB gene would be identified as a target of RNA editing in monocytes upon exposure to hypoxia or during macrophage differentiation in normoxia but not in peripheral blood. If SDHB editing aberrantly occurs in the paraganglionic tissues, an increased risk of paraganglioma development might ensue. Recent evidence suggests that tumor suppressor gene functions can be compromised by as little as a 20% decrease in gene dosage (Berger, Knudson & Pandolfi 2011). Dosage sensitivity of the SDH complex is specifically supported by the imprinted transmission of PGL tumors by SDHD mutations as paternal but not maternal transmission of SDHD mutations is known to cause highly penetrant tumors. Recent discovery of tissue-specific imprint marks in the vicinity of SDHD suggests that subtle allelic expression differences can result in discordant penetrances depending on which allele carries the mutation (Baysal et al., 2011). If programmed downregulation of SDHB by RNA editing improves monocyte survival in hypoxic environments, therapeutic manipulation of this pathway might provide a tool to modify risk in common diseases associated with macrophage infiltration. 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t rna sequence database analysis : BAM sequence alignment files of RNA-seq reads mapped against the GRCh37/hg19 human genome assembly were downloaded from public data repositories of the Illumina\u00ae BodyMap 2.03 and 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t ENCODE human transcriptome projects (Derrien et al., 2012,Tilgner et al., 2012). The two projects have analyzed poly-A+ RNA using the Illumina\u00ae HiSeq 2000 or Genome Analyzer II or IIx RNA sequencing platforms. From the BodyMap project, data for all 16 tissues from different healthy individuals was obtained. From the ENCODE project, data for 23 different cell-types/cell-lines (22 with biological duplicates) was obtained. For each BAM file, the mpileup routine in samtools7 0.1.19 (March 2013 release) was used with its default setting, (Li et al., 2009) which weighs alignment quality and ignores duplicate reads, to identify RNA-seq reads that align against chromosome 1 regions for the 843 b-long coding region of human SDHB mRNA (NCBI RefSeq8 no. NM_003000.2). The output of mpileup was then scanned with a Python 2.7 script to quantify the number of RNA-seq reads with one of five base calls (A, C, G, T or ambiguous) at each nucleotide position. The fraction of RNA-seq reads with a base call of T for c.136C was compared against the fraction with a base call of T at any of the 213 C nucleotidebearing positions of the SDHB coding region. Statistical significance of any difference was determined with two-tailed Chi-square test with Yates correction using Prism 6.0c software (GraphPad\u00ae). None of the 67 single nucleotide polymorphisms (SNPs) that have been documented for the SDHB coding region in dbSNP10 database (04-25-2012 release, NCBI dbSNP build 138 phase I) is at the genomic position of SDHB c.136C. 1 day 3 2.2% nrb : 4 day 3 8.7% 11% : a Sequencing depth ranges between 4980 and 4986 for the reported variants. b NR= Not reported. Variants detected by high-throughput sequencing are reported only when their frequency exceeds 10%. PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t Figure 1 SDHB C136U RNA editing is induced in monocytes during short-term culture. (A) C136U editing is measured in 9 lymphoblastoid cell lines and one embryonic kidney cell line, freshly isolated PBMCs (n=50), PBMCs monocyte-enriched by cold-aggregation (Mono+; n=42) and monocytedepleted PBMCs comprised of largely lymphocytes (Mono-; n=10). Horizontal lines represent mean \u00b1 standard errors throughout the figures. (B) Fraction of the C136U transcripts weakly correlates with the CD14+ monocyte percentage in monocyte-enriched PBMCs (Pearson correlation coefficient r=0.36). (C) Impact of short-term culture on C136U editing. Short-term culture of five monocyte-enriched PBMCs shows upregulation of C136U editing in adherent cells in culture days 5-7 compared to days 1-3. Day 0 represents the uncultured samples. (D) Flow cytometric sorting of CD14+ versus CD14- adherent cells on culture days 5-8 shows higher mutation rates in monocyte-macrophage lineage than in lymphocytes (p=0.04, n=5, Wilcoxon matched pairs signed ranks test). (E) C136U editing is lower among non-adherent cells in the supernatant than in adherent cells (4.3% versus 9.8%) on culture days 5-7 (n=19 wells from 11 samples on days 5-7, p=2.1 X10 -4 , Wilcoxon matched pairs signed ranks test). PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t Figure 2 SDHB C136U RNA editing correlates with adherent monocyte-rich adherent aggregates (AA) in culture. The AAs on culture days 1 (A) and 5 (B) are shown (5X low power field; vertical bars equal to 1 mm ). The culture is initiated with 30 million cells per well. The AAs grow in size until approximately days 5-7 when the C136U editing rate also usually peaks. (C) High editing rates are seen in cultures with intermediate and high density of adherent aggregates. Aggregate densities are grouped as follows: Low= less than 10 aggregates/per 5X low power field (lpf); Intermediate=10-20 aggregates/lpf; High= more than 20 aggregates/lpf (D) Giemsa stain highlights an adherent aggregate on day 6 culture. Individually attached mature macrophages have mostly round eccentric nuclei and abundant cytoplasm. Occasional multinucleated macrophages (arrowheads) and rare small lymphocytes (green arrows) are also present. Macrophages within the aggregate are smaller with less cytoplasm and occasionally have curved nuclei (arrows). (E) CD163 immunostaining of adherent aggregates. Immunocytochemical staining for monocytemacrophage-specific antigen CD163, a scavenger receptor for hemoglobin-haptoglobin complex, demonstrates that the adherent aggregates are primarily comprised of monocyte/macrophage lineage cells. Individually attached macrophages outside the aggregate also stain with variable intensity (short arrows); while numerous small lymphocytes are negative (long arrows). PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t Figure 3 SDHB C136U RNA editing increases during monocyte-macrophage maturation. Flow cytometric evaluation of monocyte-macrophage maturation is shown in uncultured cold aggregated PBMCs (first column), adherent cells on culture day 6 (second column) and adherent cells on culture day 15 (third column). The day 15 culture shows a relatively homogenous large (indicated by high forward scatter, FSC-A) macrophage population that has high complexity (indicated by high side scatter, SSC-A) and is positive for CD206 (mannose receptor), HLA-DR and CD163. In contrast, uncultured monocytes are largely a uniform population that is smaller with less complexity and is negative for CD206. The day 6 culture, which has 11.6% C136U editing rate, shows a CD14+ population that is brightly positive for HLA-DR, dim-positive for CD206 and negative for CD163. CD14+ monocyte-macrophage and lymphocyte populations are marked by green and red, respectively. PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t Figure 4 Hypoxia induces SDHB C136U RNA editing. (A) The editing rates are higher on days 1-3 in hypoxia than the average of adherent and supernatant cells in normoxic cultures (3-day average 11.2% in hypoxia versus 3.6% in normoxia; p=0.006, n=4, Wilcoxon matched pairs signed ranks test). In contrast, as the hypoxic editing rate decreases along with extensive cell death on days 5-7, it increases in normoxic cultures as previously described (Figure 1C ). The average of four independent cultures is presented. (B) C136U editing rates in 14 independent cultures in hypoxia versus normoxia during 3 days of culture are shown. The editing rates were obtained from supernatant in all hypoxic cultures (first column) and from total cell population in normoxic cultures, except in two normoxic cultures where only supernatant was collected (second column). D0 samples represent the original uncultured cold aggregated PBMCs. Hypoxia statistically significantly increased the C136U editing rates in days 1, 2 and 3. (C) PCR amplification and Taq1 restriction enzyme (RE) digestion shows no evidence of C136T DNA mutation in SDHB exon 2 genomic DNA (gDNA) (right panel) in the same samples with high C136U RNA editing rates (left panel). The C136U RNA editing rates above the lanes were measured by AS qPCR. RTand gDNA-PCRs generate amplicon sizes of 285 bp and 233 bp, respectively. Taq1 RE digestion of wild type cDNA and gDNA sequences generates 159 bp/126 bp and 131 bp/102 bp bands, respectively. C136U/T mutation destroys the RE site. (D) Western blot analysis of normoxic (N) and hypoxic (H) cultures from one donor shows no major changes in SDHB protein expression, normalized against beta actin, as the editing rate increases in hypoxia, but possibly a subtle decrease when the C136U percentage is 16.1% on day 2 in hypoxia. (E) The editing rates are higher in flow sorted CD14+ viable cells than CD14- viable cells from day 2 hypoxic cultures (average 9.35% versus 1.63%; p<0.04, n=4, Wilcoxon matched pairs signed ranks test, one-sided). In all experiments, hypoxic and control normoxic cultures derive from the same donors (i.e., oxygen tension is the only variable). PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t acknowledgments : We thank anonymous platelet donors, personnel at RPCI's core facilities for assistance with flow cytometry, high-throughput sequencing, immunocytochemistry and hypoxia treatment, Eric Kannisto for technical help, and the Department of Pathology and Laboratory Medicine for administrative and financial support. 608 609 610 611 612 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t microscopy and imaging : Standard tissue culture monitoring and imaging was performed by Zeiss Axio microscope. Adherent aggregates (approximately 100 micron or larger in diameter) were counted at low power (2.5X) magnification. Live image photographs were taken using a Leica AF6000LX system (Houston, TX) which is comprised of a Leica DMI-6000B microscope and Leica LAS AF software interface. Nucleic acid isolation and analysis RNA and DNA were isolated using Trizol (Life Technologies, Grand Island, NY) and DNA Wizard genomic DNA purification kit (Promega, Madiosn, WI), respectively. Nucleic acid lysis buffer or Trizol was added directly to the adherent cells in plate well. Nucleic acid was quantified by a spectrophotometer (Nanondrop, Thermo Scientific, Wilmington, DE). Oligonucleotide primers and templates were obtained from IDT technologies (Coralville, IA). RT and qPCR were performed following the manufacturer\u2019s kits and instructions (LightCycler 480II, Roche, Indianapolis, IN). Total RNA was synthesized using a mixture of random short oligonucleotide and oligodT primer mixture. PCR control experiments were performed to test the specificity and amplification efficiency of oligonucleotide primer pairs using synthetic oligonucleotide templates. qPCR amplification of cDNAs was performed and crossing point (Cp) levels of C136U (T assay) and total SDHB (total assay) were determined separately. Each assay was 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t performed in duplicate or triplicate wells. T assay and total assay had the same forward oligonucleotide primer in exon 1 and different oligonucleotide reverse primers in exon 2, where the C136U editing occurs. The reverse primer for the T assay had an extra T at its 3\u2019-end relative to the reverse primer in the common assay. (Oligonucleotide primers and probes used in this study are listed in supplemental Table S1.) The relative amount of the edited transcripts was calculated by the delta-Cp method by exponentiating the total transcript Cp minus the edited transcript Cp value to the power of 2. This approach assumes 100% duplication efficiency per cycle in both assays. Control PCR reactions were performed to test the specificity and amplification efficiency of oligonucleotide primer pairs using synthetic oligonucleotide templates. Assays with 8-fold serially diluted synthetic oligonucleotide templates for the wild-type and C136T edited sequences showed a duplication efficiency of 107% for both total and T assays between 1 femtomole and 0.245 attomole (Supplemental Table S2). The mutation specific primer had a high specificity for the edited transcripts, with an average false positive amplification rate of 1.38% when the control wild type oligonucleotide template sequence was amplified in an over 25, 000 fold concentration range. When 100% T template was used, the average estimate of the C136U edited transcripts was 91% between 8 femtomoles and 0.245 attomoles concentration range. These control experiments suggest that AS qPCR estimates may be slightly lower than the true editing rates. We chose this stringent AS qPCR method to ensure we did not overestimate the C136U editing rates. The estimated percentage of edited transcripts using this method was highly reproducible in biological samples. For example, a set of 14 samples containing both low and high levels of edited transcripts gave very similar results in two replicate experiments with a Pearson correlation coefficient of 99.4%. In each assay, a positive control sample which had a mutation rate of 15-17% was included. Relative quantification of CDA and SDHB expression was 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t performed by 2^(-delta delta(Ct)) method (Livak & Schmittgen 2001) using beta-2 microglobulin as the control housekeeping gene. Microarray analysis of gene expression Expression profiling was accomplished using the Human HT-12 whole-genome gene expression array and direct hybridization assay (Illumina, Inc.). Initially, 500 ng total RNA was converted to cDNA, followed by in vitro transcription to generate biotin labeled cRNA using the Ambion Illumina TotalPrep RNA Amplification Kit (Ambion, Inc.) as per manufacturer\u2019s instructions. 750 ng of the labeled probes were then mixed with hybridization reagents and hybridized overnight at 58 \u00b0C to the HT-12v4 BeadChips. Following washing and staining with Cy3-streptavidin conjugate, the BeadChips were imaged using the Illumina iScan Reader to measure fluorescence intensity at each probe. The intensity of the signal corresponds to the quantity of the respective mRNA in the original sample. The background corrected gene expression levels were extracted from BeadChip using Illumina\u2019s Genome Studio (v2011.1) gene expression module (v1.9.0). The log2 transformed expression levels were quantile normalized using Lumi module in the R-based Bioconductor package (Gentleman et al., 2004). For data quality control, we excluded the genes with detection p-value greater than 0.05 (i.e., indistinguishable from the background noise). 18941 out of 34686 genes passed this filtering for downstream analysis. The Limma program (Smyth 2004) was used to calculate the level of differential gene expression. Briefly, a linear model was fit to the data (paired design, with cell means corresponding to the different condition and a random effect for array) and selected contrasts of condition (i.e., case vs. control) were performed. A list of differentially expressed genes with P <0.05 and \u22652 fold-change was obtained and analyzed for enriched Gene Ontology (GO) categories and KEGG pathways using 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t NCBI DAVID Bioinformatics Resources (Huang da,Sherman & Lempicki 2009). The enriched GO terms and KEGG pathways with P <0.05 and \u22655 genes were kept. accession number : Microarray data have been deposited in GEO (www.ncbi.nlm.nih.gov/geo/) under accession number GSE45900. High-throughput RT-PCR amplicon sequencing SDHB and SDHD gene transcripts were amplified with oligonucleotide primers derived from 5\u2019- and 3\u2019-UTR using Roche reverse transcriptase and Pfu Ultra2 (Agilent, Santa Clara, CA) high fidelity PCR amplification. Library preparation was performed using the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA). High-throughput sequencing is performed on a MiSeq personal sequencing platform. flow cytometry : Cells (0.5-1x106) were pelleted by centrifugation at 400 x g for 5 minutes and incubated for 20 minutes at room temperature in a total volume of 100 \u00b5l with a titrated cocktail of APC-conjugated CD14 (Invitrogen, Carlsbad, CA), Brilliant-violet 421 conjugated CD16 (Biolegend, San Diego, CA), PE-conjugated CD163 (Trillium Diagnostics, Brewer, ME), FITC-conjugated CD206 (BD Biosciences, San Jose, CA), and PE-Cy7-conjugated HLA-DR (BD Biosciences, San Jose, CA), followed by a wash in PBS and resuspension in 500 \u00b5l PBS. Flow cytometric acquisition was performed on a FACS Aria I flow cytometer (BD Biosciences) equipped with four laser excitation sources (405 nm 30 mW; 488 nm 15 mW; 561 nm 30 mW; and 631 nm 17 mW) that was qualitycontrolled on a daily basis by using CS&T beads and FACS DiVa software (BD Biosciences). The filter configurations for the PMTs measuring fluorescence emission of the applied fluorochromes were 450/50 nm (Brilliant-violet 421); 530/30 nm (FITC); 582/15 nm (PE); 780/60 nm (PE-Cy7); 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t and 660/20 nm (APC). Autofluorescence and single-color controls were acquired to perform spectral overlap compensation using the automated compensation matrix feature in FACS DiVa software. A forward scatter threshold was applied to eliminate electronic noise and small particles from the flow cytometric acquisition. A target of 20,000 scatter-inclusive events was acquired for each specimen. Data analysis was performed with FlowJo software version 10.0.4 (Tree Star, Inc., Ashland, OR). isolation of cd14+ monocytes : Monocytes were isolated either by flow cytometric gating of CD14+ (both dim and strong intensity) or by CD14 microbeads (Miltenyi Biotec Inc. Auburn, CA) following manufacture\u2019s protocol. Flow cytometry showed 93% or higher purity after CD14 microbead purification. immunocytochemical staining : PBMCs were grown in tissue culture chamber slides (Lab Tek Chamber slide, Fisher Scientific). Before staining, supernatant was discarded and adherent cells were washed once with PBS. Adherent cells were fixed by alcohol-based cytology fixative (Leica microsystems) and air dried. For antigen retrieval, slides were heated in the steamer for 20 minutes in citrate buffer (pH 6.0), followed by a 20 minute cool down. Endogenous peroxidase was quenched with aqueous 3% H2O2 for 10 minutes and washed with PBS/T. Slides were loaded on a DAKO autostainer and a serum free protein block (Dako catalog #X0909) was applied for 5 minutes, blown off, and the antibody applied for one-hour. Biotinylated goat anti-mouse IgG (Jackson Immuno Research Labs, catalog #115-065-062) was applied for 30 minutes, followed by the Elite ABC Kit (Vectastain) for 30 minutes, and the DAB chromagen (Dako) for 5 minutes. Finally, the slides were counterstained with hematoxylin, dehydrated, cleared and cover slipped. Western blot analysis 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t Cell lysates were prepared using 75 \u03bcl of M-PER protein extraction reagent (Thermo, Waltham, MA) per approximately 20X106 washed cells. The concentration of proteins in lysates was determined with the colorimetric Pierce 660 nm protein assay (Thermo). For electrophoresis, lysates (40\u201360 \u03bcg of protein) were boiled for 5 min in buffer containing 50 mM Tris, 2% SDS and 143 mm \u03b2-mercaptoethanol. After electrophoresis in Mini-PROTEAN\u2122 TGX precast 4%\u201315% gradient polyacrylamide gels (Bio-Rad, Hercules, CA) at 15\u201330 mA for 90 min, proteins were transferred overnight at 30 mA in 10% methanol-containing Tris-glycine buffer to 0.45 \u00b5 polyvinylidene difluoride membrane (GE Healthcare, Little Chalfont, UK). For immunoblotting, membranes were blocked in Tris-buffered saline (TBS; 10 mM Tris-HCl and 150 mM NaCl at pH 7.4) with 0.05% Tween-20 (Sigma, Saint Louis, MO) and 5% non-fat milk (Carnation\u2122, Nestle), and then incubated in the same solution with a primary mouse monoclonal anti-SDHB antibody (sc-271548, Santa Cruz Biotechnology, Dallas, TX) at room temperature for an hour. After washing in TBS/Tween-20 for 30 minutes, membranes were incubated in TBS/Tween20/milk with a horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. Membranes were then washed in TBS/Tween-20 for 30 min, incubated in Luminata Forte\u2122 chemiluminescence reagent (Thermo), and exposed to photographic film (Thermo). Mouse anti-beta-actin (clone C4, Santa Cruz Biotechnology) and horseradish peroxidase-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA) antibodies were used at 1:200 and 1:2500 dilutions, respectively. Band intensities were determined by a densitometer. the average of two density measurements from beta actin was used to determine relative amounts of SDHB protein. c136u editing rate increases during short-term culture of pbmcs in a time-dependent manner : We cultured monocyte-enriched PBMCs for in vitro macrophage differentiation by plate adherence in the presence of 10% fetal bovine serum (FBS) at standard culture conditions as described (Roiniotis et al., 2009). Flow cytometry showed that the fraction of CD14+ cells among the adherent cells on culture days 5-6 (25.9% \u00b1 5.2%; n=4) was comparable to the uncultured monocyte-enriched PBMC samples (27.2%\u00b1 2.7%; n=10). Initial cultures showed that the fraction of the edited transcripts increased in the adherent cells on days 5-7 then decreased to baseline levels on later days. (Supplemental Figure S1A). Short-term cultures showed that the editing rates were lower in the first three days than on days 5-7 (Figure 1C , 3.54% versus 11.62%, n=15, p=8X10-4, Wilcoxon matched pairs signed ranks test). The uncultured monocyte-enriched PBMC samples had lower C136U editing rates than their matched cultures on days 5 and 6 in 17 of the 17 samples (1.7% \u00b1 0.2% versus 10.06% \u00b1 0.8%; p=2.9X10-4, Wilcoxon matched pairs signed ranks test). To confirm monocyte origin of the edited transcripts in the cultured samples, we performed flow cytometric sorting of the attached CD14+ cells from culture days 5-8 which essentially separated monocyte/macrophages from lymphocytes to >95% purity. In five of five samples, the CD14+ cells showed higher editing rates than the CD14- cells (Figure 1D). The editing rate was higher among the adherent cells than those in the supernatant (Figure 1E). Collectively, these findings indicate that C136U editing is induced in monocyte/macrophage lineage cells during culture of monocyte-enriched PBMCs in a timedependent manner. The maximum mutation rate reached on days 4-8 varied markedly among samples from 1.2% to 18.8%. Total number of cultured cells per well correlated positively but very weakly with the maximum mutation rate (Supplemental Figure S1B). The maximum C136U editing rate trended higher when the total number of monocyte-enriched PBMCs was over 21 million per well versus 20 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t million or less per well, though this difference did not reach statistical significance (10.9% \u00b1 1.23%; n=16 versus 8.54% \u00b1 1.03%; n=15; p=0.1; Mann Whitney U test, one-sided). Morphologic evaluation of cultures associated with C136U editing We noted the development of three dimensional adherent aggregates (AAs) in monocyte-enriched PBMC cultures, especially in those that had the highest C136U editing rates. These aggregates were detectable within 12 hours and increased in density and size up to a week in culture (Figure 2A and 2B). Hourly photographic imaging in the first two days showed that the aggregates aggregates start as a loose collection of cells and become larger and more compact by merging and recruitment of individual cells (Supplemental video S1). Cultures with low AA densities showed lower mutation rates than those with intermediate or high AA densities (p=0.015, n=16, Mann-Whitney U test, Figure 2C). Morphologic examination showed that the AAs were primarily comprised of intermediate-size cells that generally had round non-convoluted nuclei and a moderate amount of cytoplasm. Occasional lymphocytes and large monocytoid cells with curved nuclei were also noted within the aggregates. In contrast, numerous individually attached macrophages, including those with spindle-like morphology or multi-nucleation, had more abundant cytoplasm compared to cells in the AAs (Figure 2D). Immunostaining with CD163 antibody confirmed that most cells in the AAs were aggregated monocytes/macrophages (Figure 2E). These results suggest that C136U editing primarily occurs in aggregating monocytes that are in the process of differentiating to macrophages. It is conceivable that micro-environmental nutrient and/or oxygen depravation associated with the dense adherent aggregates of monocytes may induce SDHB RNA editing. 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t Microarray analysis of differentially expressed genes associated with increased C136U editing in normoxia We performed pairwise microarray gene expression analysis of four sets of low-editing (day 3) and high-editing (day 5-8) samples cultured in normoxia (Table 1) to evaluate (a) global changes in cellular pathways; (b) whether adherent aggregates are associated with micro-environmental hypoxia; and (c) changes in the SDH subunit and cytidine deaminase family of genes. Analysis showed that 171 genes were upregulated and 123 genes were downregulated genes by at least twofold at a statistically significant level (P <0.05). The subset of these genes (n=55) that showed \u2265 3 fold-change is shown in Supplemental Table S3. The complete list is available on GEO database under accession number GSE45900. No SDH subunit or APOBEC family gene met these criteria. Cytidine deaminase (CDA) was the only gene from the cytidine deaminase family of genes that had its expression level changed significantly, with a 3.1-fold increase. qPCR analysis confirmed upregulation of CDA expression (average 3.7-fold) and little change in SDHB expression (average 0.98-fold) in the high-editing samples relative to the low-editing ones. Significant gene expression changes coordinately occurred in functionally related genes in several Gene Ontology (GO) categories (n=159). This number decreased to 10 after Bonferroni correction for multiple testing was applied (Supplemental Table S4). These 10 categories included defense to wounding, inflammatory, immune and defense responses, taxis, chemotaxis, carboxylic and organic acid transport, locomotory behavior and regulation of cell proliferation. Notably, hypoxia-response genes were not categorically different between the low and the high editing samples. These results indicate that significant gene expression changes occur in inflammatory and immune response pathways during increase in SDHB editing and that CDA is a candidate gene for enzymatic C136U deamination. Flow cytometric characterization of cultures associated with C136U editing 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t To evaluate monocyte macrophage maturation in PBMC culture, we performed flow cytometric characterization of the attached cells using monocyte/macrophage-associated antibodies for CD14, CD16, CD163, HLA-DR and CD206, a mannose receptor associated with M2 type macrophage polarization that promotes wound healing (Porcheray et al., 2005). Cold-aggregated uncultured PBMCs showed a discrete and relatively uniform monocytic population that is positive for CD14, HLA-DR and CD163; but negative for CD206 and largely negative for CD16 (Figure 3). Cultured adherent cells at day 6, which showed an increased editing rate, contained a CD14+ population comprised of larger cells with more complexity and were dimpositive for CD206, heterogeneously positive for CD14 and brightly positive for HLA-DR. CD16 and CD163 patterns were similar and showed a heterogeneous negative-to-positive range. Analysis of the adherent cells at day 15, when the editing rates are typically low and the AAs are loose (Supplemental Figure S1C), showed a mature macrophage population that had increased complexity and a more uniform antigenic profile, including homogenous positivity for CD14, CD16, CD163, HLA-DR and CD206. Taken together, these results demonstrate that high C136U editing peaks during monocyte macrophage differentiation in normoxic culture. When monocyte-enriched PBMC cultures were treated with m-CSF and GM-CSF/IL4 to facilitate macrophage and dendritic cell differentiation, respectively, lower editing rates were seen compared to the control cultures that contained only 10% FBS (p=0.012, n=8 and p=0.04, n=5, respectively, Wilcoxon matched pairs signed ranks test Supplemental Figure S1D). Similarly, lipopolysaccharide (LPS) treatment of monocyte-enriched PBMCs minimally but statistically significantly reduced the editing rates during the first three days of culture (0.62% in LPS-treated group versus 1.17% in 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t controls in the total cell population [n=4]; p=0.013, Wilcoxon matched pairs signed ranks test). Notably, LPS and cytokine-treatment, especially with GM-CSF/IL4, also diminished adherent aggregate formation under normoxia. Next, we tested C136U editing rates in the monocytic leukemia cell line THP-1. We added macrophage differentiation agents to the media with 10% FBS including phorbol ester PMA (4 beta-phorbol 12-myristate 13-acetate), retinoic acid and vitamin D. Significant C136U editing was not identified (less than 1%) either in the untreated THP-1 cell line or after treatment with the differentiating agents. Similarly, hypoxia exposure of the THP-1 cell line showed no increase in C136U levels in days 1, 2 and 3 (less than 1%). These results suggest that C136U editing under normoxic culture conditions primarily occurs in mature monocytes within the dense adherent aggregates during differentiation to macrophages in the presence of serum and that LPSdriven monocyte activation (Rossol et al., 2011) or cytokine-driven macrophage or dendritic cell differentiation or leukemic transformation reduces the editing rates. Induction of C136U editing by hypoxia We evaluated the role of hypoxia on C136U editing for the following reasons. First, tissues with inflammation and infarction harboring monocyte/macrophage lineage cells are often hypoxic (Riboldi et al., 2013). Subsequently, monocyte-macrophage differentiation frequently occurs in tissues where oxygen pressure is lower than that in blood. Second, the dense adherent aggregates associated with increased SDHB editing in normoxic cultures might conceivably be associated with micro-environmental hypoxia. We observed that monocyte-enriched PBMC cultures in hypoxia did not develop the firmly adherent aggregates associated with high editing rates in the normoxic cultures and that essentially all cells were in suspension. Fraction of viable CD14+ monocytes among total cells in the hypoxic supernatants on culture day 2 was similar to the original uncultured samples as evaluated by flow cytometry (20.9% versus 20.4%, n=4). The editing rate in the hypoxic cultures increased in the first three days with a peak on day 2, when it is typically low in the 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t normoxic control cultures (Figure 1C and 4A). In five of the 14 samples, C136U editing rates were higher (21%-49%) than in any normoxic culture. Hypoxia increased the fraction of C136U transcripts by 0.4% to 46% on culture day 2. The variable increase in the percentage of C136U transcripts could be caused by technical or biological factors including variable fraction of monocytes in donors, variable enrichment of monocytes by the cold-aggregation method, variation in inherent enzymatic activity of the putative editing enzyme or nonsense mediated decay pathway activation.(Chang,Imam & Wilkinson 2007). Pairwise comparison of the editing rates in supernatant cells in hypoxia versus total cells in normoxia from 14 different samples showed marked upregulation of C136U editing by hypoxia in the first three days (p=2X10-7, Wilcoxon matched pairs test; Fig. 4B). Hypoxia increased C136U fractions in 39 of 42 pairwise comparisons from 14 samples cultured in normoxia and hypoxia for three days. Comparison of the flow sorted CD14+ and CD14\u2013 cell populations confirmed that monocytes were the main source of C136U editing in hypoxia (Figure 4E). Hypoxic culture of granulocytes obtained from two donors, plated at 0.6 and 1.2 million cells/ml densities, respectively, showed no evidence of C136U editing (0.89% in normoxic and hypoxic granulocytes; n=2). In contrast, monocyte-enriched PBMCs from the same donors showed very high editing rates (>45%) under hypoxia. These findings indicate that hypoxia-induced C136U SDHB RNA editing occurs primarily in monocytes among peripheral blood leukocytes. RT-qPCR analysis showed marked upregulation of CDA (average 7.5-fold change on day 2) and downregulation of SDHB (average 0.37-fold change on day 3) in the hypoxic cultures relative to the control gene beta 2 microglobulin. Western blot analysis of one monocyte-enriched PBMC sample cultured in normoxic and hypoxic conditions showed no marked changes in the expression SDHB protein product (Figure 4D), suggesting that the impact of C136U editing on protein expression levels is subtle and quantitative. 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t Analysis of genomic DNA for C136T editing To test whether the C136U RNA mutation has a corresponding C-to-T genomic DNA mutation in SDHB exon 2, we compared mutant RNA and DNA levels using our previously described semiquantitative method based on PCR amplification and Taq1 restriction enzyme digestion (Baysal 2007). We found no evidence of a corresponding DNA mutation in the hypoxic samples containing high levels of C136U RNA mutations (Figure 4C). These results indicate that C136U mRNA editing occurs during or after transcription. High throughput sequencing of SDHB and SDHD transcripts To obtain a global view of transcript editing in the SDHB and the SDHD genes, we performed high throughput sequencing of the full length coding transcripts obtained by RT and high-fidelity PCR. We sequenced the four pairs of normoxic cultures that were characterized by microarray gene expression analyses. Each pair derived from the same PBMC and contained a sample with a low C136U editing rate (day 3 culture) and a sample with a high C136U editing rate (day 5-8 culture). Interrogation of the transcript sequences confirmed C136U mutations in each of the high-editing-rate sample. On average, 40% higher editing levels were seen by high throughput sequencing than those estimated by AS qPCR (Table 1). No additional canonical RNA edits in the form of A-to-I/G or Cto-U/T changes were identified in the SDHB transcripts. No non-canonical SDHB transcript variants could be confirmed by Sanger sequencing. In contrast, Sanger sequencing confirmed C136U in all four samples that showed high editing rates by AS qPCR and by high-throughput sequencing (Supplemental Figure S1E). Similarly, SDHD transcripts showed no evidence of RNA editing by high-throughput sequencing. Taken together, these results demonstrate that SDHB C136U editing is not accompanied by other transcript variants either in SDHB or SDHD and suggest that a site-specific C-to-U RNA editing mechanism regulates the SDHB functional transcript dosage. Analysis of transcriptome sequence databases 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t We examined RNA sequencing data of the Illumina Human Body Map and ENCODE Human Transcriptome projects (Derrien et al., 2012, Tilgner et al., 2012, Brazma et al., 2003) for sequence variations in the ORF region SDHB mRNA. As expected, among the 16 normal human tissues from Human Body Map, the highest levels of SDHB C136U editing was seen in white blood cells (1.7%), with the other 15 tissues showing an average editing level of 0.2% (SD=0.3%), with ovary (1%) and kidney (0.9%) being those with the highest levels among the 15 (Supplemental Table S5). Among the 45 samples of 23 different cell-types of the ENCODE set, the editing rate was highest for the two primary CD14+ monocytes (1.9% and 2.6%), with the other samples showing an average 0.1% (SD=0.2%) editing rate (Supplemental Table S6). The editing rates noted for the samples for white blood cells and the monocytes are unlikely to be a result of sequencing error since a base call of U instead of C was made in only 0.04%-0.09% of the reads when all 213 C-bearing positions of the SDHB coding region were examined (Chi square test, P <0.001). These results support our earlier conclusions that C136U editing occurs at low but statistically significant levels in peripheral blood monocytes. authorship : BEB conceptualized the study and wrote the manuscript. BEB, RT, JW and PKW designed experiments. RT, KDJ and BEB performed experiments. BL and JW performed the bioinformatic analysis of microarray expression data. SP performed the ENCODE and Illumina database analysis. All authors approved the final manuscript. 603 604 605 606 607 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t li h, handsaker b, wysoker a, fennell t, ruan j, homer n, marth g, abecasis g, durbin : R, 1000 Genome Project Data Processing Subgroup. 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics (Oxford, England) 25: 2078. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif.) 25: 402. Lopez-Jimenez E, Gomez-Lopez G, Leandro-Garcia LJ, Munoz I, Schiavi F, MonteroConde C, de Cubas AA, Ramires R, Landa I, Leskela S, Maliszewska A, Inglada-Perez L, de la Vega L, Rodriguez-Antona C, Leton R, Bernal C, de Campos JM, Diez-Tascon C, Fraga MF, Boullosa C, Pisano DG, Opocher G, Robledo M, Cascon A. 2010. 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Last accessed 8/9/2013. 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t supplemental material : Supplemental Figure S1 Impact of long-term culture and cell density on C136U editing are shown. (A) Five monocyte-enriched PBMCs were cultured long-term and the C136U editing rates were measured. The editing rate was higher in adherent cells on days 5-7 than in the uncultured monocyteenriched PBMCs and on days 12-14 and 19-21 (p=0.017, n=5, one-way ANOVA). (B) Maximum C136U editing rate in the adherent cells shows very weakly positive correlation with the total number of monocyte-enriched PBMCs per well. Total number of samples=31. (C) Attached aggregates (AAs) on culture day 20 are shown. The AAs start loosening after one week in culture and eventually appear as flat areas of increased cellular density. This culture was fed with fresh RPMI-1640/10% FBS on day 8. (D) Cytokine mediated differentiation towards macrophages or dendritic cells reduce C136U editing rates. Cultures containing 10% FCS showed higher mutation rates than those treated with M-CSF (p=0.012) or GM-CSF/IL4 (p=0.04). Multi-day averages from two PBMC cultures are shown. Matched samples were collected on days 4-8 for the m-CSF culture (n=8) and days 4-6 for the GMCSF/IL4 cultures (n=5). The editing rate was obtained from adherent cells in 10% FCS and m-CSF treated (macrophage-differentiation) wells and from non-adherent cells in supernatant in GMCSF/IL4-treated (dendritic cell-differentiation) wells. (E) Sanger sequencing confirms C136U RNA editing in all four samples that showed high editing rates by RT-qPCR and high-throughput sequencing. Chromatograms show examples of a sample with a low editing rate (sample 1, day3 in Table 1) and one with a high editing rate (sample 4, day 7 in Table 1). C136U variant is shown by arrows. 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t Supplemental Video S1 Culture is initiated after mild monocyte enrichment by cold aggregation. After 5 hours the supernatant is removed and hourly photographic imaging is started. Video is a composite of sequential images (time-lapse) for the first 48 hours. Note that the aggregates start as a loose collection of cells and become larger and more compact by cell recruitment. 757 758 759 760 PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t Table 1(on next page) Percentage of C136U transcripts estimated by allele specific (AS) qPCR and high-throughput amplicon sequencing in normoxic cultures PeerJ reviewing PDF | (v2013:06:588:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t Table 1 Percentage of C136U transcripts estimated by allele specific (AS) qPCR and high-throughput amplicon sequencing in normoxic cultures Sample No. Day in culture AS qPCR high-throughput sequencinga",
    "v2_text": "materials and methods : PBMC isolation and cell culture Leukocytes were isolated from Trima leukoreduction filters (Terumo BCT, Lakewood, CO) of anonymous healthy platelet donors following an IRB review and determination as non-human subject research. PBMCs were purified by histopaque 1077 (Sigma-Aldrich, St. Louis, MO) and washed twice with RPMI-1640/10% fetal bovine serum (FBS) to remove residual platelets. Each filter often gave 5X108 or more PBMCs. Monocytes were enriched by the cold aggregation method (Mentzer, et al., 1986) with certain modifications. PBMCs were suspended in 35 ml RPMI-1640 in a 50 ml polypropylene tube and incubated at 4 oC for 1 h on a rocker panel for homotypic aggregation of monocytes. For monocyte enrichment, the tube was positioned upright, underlaid with 6 ml FBS and incubated overnight at 4 oC. The cell precipitate under the serum was collected for culture. Monocyte enrichment was approximately 70% (n=5) by short-term precipitation (up to 3 hours) and 27% (n=10) by overnight precipitation over serum as determined by percentages of the CD14+ cells by flow cytometry. The non-aggregating upper layer was markedly depleted of monocytes (less than 4%, n=3). We used the overnight precipitation method which gave higher yield of total cells and were associated with higher levels of C136U editing in culture. Precipitated cells under the serum were centrifuged for 10 min at 250xg and suspended in 20 ml RPMI-1640 with 10% FBS and penicillin/streptomycin. Cell density was calculated by a hemocytometer (improved Neubauer , InCyto, Covington, GA). After further dilution, two milliliters of the cell suspension was distributed to each well of a six-well tissue culture plate (Costar, Corning Incorporated, Corning, NY). Editing rates were determined in daily collected individual wells. We cultured 21-30x106 cells/2 ml per well (also see results). Cultures were incubated at, 5% CO2 with either 21% O2 (normoxia) or 1% O2 combined with 94% nitrogen (hypoxia). Hypoxic cultures were pre-incubated at 37 oC, 21% O2 for 4 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t approximately 3 hours before being placed in the hypoxia chamber (XVIVO system, BioSpherix, Lacona, NY). Fresh culture media was added after 7 days in culture. The THP-1 monocytic leukemia cell line was purchased from ATCC (Manassas, VA). Various cytokines/differentiating agents were added for the following final concentrations: macrophage colony stimulating factor (M-CSF; 50 ng/ml), granulocyte-macrophage (GM)-CSF (50 ng/ml), interleukin 4 (IL4; 50 ng/ml, vitamin D (10 nM), retinoic acid (1 \u00b5M), PMA (4-beta-phorbol-12-myristate-13-acetate; 100 nM). Cytokines were purchased from Peprotech (Rocky Hill, NJ) and differentiating agents were purchased from Sigma-Aldrich. results and discussion : Analysis of C136U mutation rate in PBMCs by RT and AS qPCR To determine the prevalence, distribution, cell type origin and other factors influencing SDHB editing, we developed a highly specific and reproducible assay for allele specific quantitative PCR amplification (AS qPCR) of the total and edited C136U SDHB mRNAs (see methods). Using this assay, we found that the C136U transcripts were very low or absent in B cell lymphoblastoid and fibroblastic cell lines. The editing occurred at slightly higher levels in PBMCs isolated from fresh blood than in the cell lines (Figure 1A). To study the relative contribution of monocytes and lymphocytes to SDHB editing, we performed monocyte enrichment using the well-established cold aggregation method (Mentzer, et al., 1986) with certain modifications (see methods). This method essentially separates PBMCs into cold-aggregating monocyte-enriched and non-aggregating monocyte-depleted (i.e., predominantly lymphocytic) compartments. Monocyte-enriched samples by the cold aggregation method showed higher editing rates than the matched PBMCs (2.16% versus 1.48%, n=36, P=1.6X10-5, Wilcoxon matched pairs signed ranks test) and than the matched monocyte-depleted samples (1.47% versus 0.58%, n=10, P=0.005, Wilcoxon matched pairs signed ranks test) (Figure 1A). To further confirm monocyte-origin of the edited transcripts, we isolated peripheral blood monocytes to >92% purity by CD14+ microbeads and tested the mutation rates in three samples. In each sample, a higher mutation rate was found in the monocytes than the CD14lymphocytes (average 0.84% vs 0.38%). A positive but weak correlation was found between the CD14+ monocyte-fraction and the C136U editing rate in cold-aggregated PBMC samples (Figure 1B). These results indicate that C136U editing occurs in freshly isolated monocytes in low but statistically significantly higher rates than in lymphocytes. 13 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t figure legends : Figure 1 SDHB C136U RNA editing is induced in monocytes during short-term culture. (A) C136U editing is measured in 9 lymphoblastoid cell lines and one fibroblastic cell line, freshly isolated PBMCs (n=50), PBMCs monocyte-enriched by cold-aggregation (Mono+; n=42) and monocyte-depleted PBMCs comprised of largely lymphocytes (Mono-; n=10). Horizontal lines represent mean \u00b1 standard errors throughout the figures. (B) Fraction of the C136U transcripts weakly correlates with the CD14+ monocyte percentage in monocyte-enriched PBMCs (Pearson correlation coefficient r=0.36). (C) Impact of short-term culture on C136U editing. Short-term culture of five monocyte-enriched PBMCs shows upregulation of C136U editing in adherent cells in culture days 5-7 compared to days 1-3. Day 0 represents the uncultured samples. (D) Flow cytometric sorting of CD14+ versus CD14- adherent cells on culture days 5-8 shows higher mutation rates in monocyte-macrophage lineage than in lymphocytes (P=0.04, n=5, Wilcoxon matched pairs signed ranks test). (E) C136U editing is lower among non-adherent cells in the supernatant than in adherent cells (4.3% versus 9.8%) on culture days 5-7 (n=19 wells from 11 samples on days 5-7, P=2.1 X10-4, Wilcoxon matched pairs signed ranks test). 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 R ev ie w in g M an us cr ip t highlights an adherent aggregate on day 6 culture. Individually attached mature macrophages have mostly round eccentric nuclei and abundant cytoplasm. Occasional multi-nucleated macrophages (arrowheads) and rare small lymphocytes (green arrows) are also present. Macrophages within the aggregate are smaller with less cytoplasm and occasionally have curved nuclei (arrows). (E) CD163 immunostaining of adherent aggregates. Immunocytochemical staining for monocyte-macrophage-specific antigen CD163, a scavenger receptor for hemoglobin-haptoglobin complex, demonstrates that the adherent aggregates are primarily comprised of monocyte/macrophage lineage cells. Individually attached macrophages outside the aggregate also stain with variable intensity (short arrows); while numerous small lymphocytes are negative (long arrows). 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 R ev ie w in g M an us cr ip t 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 R ev ie w in g M an us cr ip t statistical analysis : Unless specified otherwise, statistical analyses of experimental data was performed using the online interface of SISA at http://www.quantitativeskills.com/sisae (Uitenbroek, 2013) using two-tailed non-parametric tests, graphics and descriptive statistics were generated in Excel (version 10; Microsoft, Redmond, WA). Means and standard errors are depicted by horizontal bars in the figures. 12 237 238 239 240 241 242 243 244 245 246 247 248 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t conclusions : We found that an acquired RNA nonsense mutation (R46X) introduced by C-to-U RNA editing dynamically reduces the SDHB functional transcript dosage in monocytes during early macrophage differentiation and upon short-term exposure to hypoxia. The fraction of C136U transcripts was variable in monocyte-enriched PBMC cultures and peaked around 19% in normoxic cultures but increased to up to 49% within 2 days in hypoxia. The editing rates must be higher in pure monocytes because CD14+ cell isolation increased the percentages of C136U transcripts by 1.25-fold in normoxic cultures and 1.68-fold in hypoxic cultures. Gene expression analyses identified CDA as a candidate gene for SDHB editing. To our knowledge, these results provide the first examples of hypoxia-inducible coding RNA editing and a programmed mutation targeting an endogenous nuclear gene in myeloid cells. On the basis of genetic studies on SDH-mutated paragangliomas that show constitutive activation of hypoxia-inducible pathways, we hypothesize that reduction in the SDHB dosage by RNA editing facilitates hypoxia adaptation in monocytes by amplifying hypoxia signaling from mitochondria. We speculate that the programmed SDHB RNA editing during normoxic macrophage differentiation might serve to augment signaling of inflammatory hypoxia. Although the precise mechanisms linking hypoxia sensing and signaling to SDHB RNA editing remain to be determined, parallels can 21 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t be drawn between the monocytes circulating in the high-oxygen environment of the peripheral blood and subsequently migrating into the hypoxic areas where differentiation into macrophages occurs versus certain facultatively aerobic organisms such as the intestinal parasitic worm Ascaris suum (A. suum). A. suum spores living in open air are exposed to high oxygen concentrations and utilize SDH; whereas parasitic adult worms living in the hypoxic environment of intestine use fumarate reductase (Kita, et al., 2002). Thus, suppression of SDH might be an evolutionarily conserved metabolic adaptation to hypoxia achieved by diverse molecular mechanisms and might contribute to increased monocyte/macrophage survival observed under hypoxia due to enhanced glycolysis (Roiniotis, et al., 2009). More broadly, our results extend previous observations associating external factors with differential A-to-I RNA editing (Garrett & Rosenthal 2012; Balik, et al., 2013; Sanjana, et al., 2012) and demonstrate in a robust experimental model that C-to-U coding RNA editing of certain genes occurs dynamically in a cell type and environment-dependent manner. Our findings suggest that gene targets of RNA editing could be missed by whole transcriptome sequence analyses unless tissues are examined in their physiologically relevant different states. If the threshold for significant RNA editing is set at 10% or more in whole transcriptome sequence analyses (for example in Park, et al., 2012), the SDHB gene would be identified as a target of RNA editing in monocytes only upon exposure to hypoxia or during macrophage differentiation but not in peripheral blood. If SDHB editing aberrantly occurs in the paraganglionic tissues, an increased risk of paraganglioma development might ensue. Recent evidence suggests that tumor suppressor gene functions can be compromised by as little as a 20% decrease in gene dosage (Berger, Knudson & Pandolfi 2011). Dosage sensitivity of the SDH complex is specifically supported by the imprinted transmission of PGL tumors by SDHD mutations as paternal but not maternal transmission of SDHD mutations is 22 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t known to cause highly penetrant tumors. Recent discovery of tissue-specific imprint marks in the vicinity of SDHD suggests that subtle allelic expression differences can result in discordant penetrances depending on which allele carries the mutation (Baysal, et al., 2011). If programmed downregulation of SDHB by RNA editing improves monocyte survival in hypoxic environments, therapeutic manipulation of this pathway might provide a tool to modify risk in common diseases associated with macrophage infiltration. 23 472 473 474 475 476 477 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t acknowledgments : We thank anonymous platelet donors, personnel at RPCI's core facilities for assistance with flow cytometry, high-throughput sequencing, immunocytochemistry and hypoxia treatment, Eric Kannisto for technical help, and the Department of Pathology and Laboratory Medicine for administrative and financial support. rna sequence database analysis : BAM sequence alignment files of RNA-seq reads mapped against the GRCh37/hg19 human genome assembly were downloaded from public data repositories of the Illumina\u00ae BodyMap 2.03 and ENCODE human transcriptome projects (Derrien, et al., 2012,Tilgner, et al., 2012). The two projects have analyzed poly-A+ RNA using the Illumina\u00ae HiSeq 2000 or Genome Analyzer II or IIx RNA sequencing platforms. From the BodyMap project, data for all 16 tissues from different healthy individuals was obtained. From the ENCODE project, data for 23 different cell-types/cell-lines (22 with biological duplicates) was obtained. For each BAM file, the mpileup routine in samtools7 0.1.19 (March 2013 release) was used with its default setting, (Li, et al., 2009) which weighs alignment quality and ignores duplicate reads, to identify RNA-seq reads that align against chromosome 1 regions for the 843 b-long coding region of human SDHB mRNA (NCBI RefSeq8 no. NM_003000.2). The output of mpileup was then scanned with a Python 2.7 script to quantify the number of RNA-seq reads with one of five base calls (A, C, G, T or ambiguous) at each nucleotide position. The fraction of RNA-seq reads with a base call of T 11 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t for c.136C was compared against the fraction with a base call of T at any of the 213 C nucleotide-bearing positions of the SDHB coding region. Statistical significance of any difference was determined with two-tailed Chi-square test with Yates correction using Prism 6.0c software (GraphPad, La Jolla, CA). None of the 67 single nucleotide polymorphisms (SNPs) that have been documented for the SDHB coding region in dbSNP10 database (04-25-2012 release, NCBI dbSNP build 138 phase I) is at the genomic position of SDHB c.136C. western blot analysis : Cell lysates were prepared using 75 \u03bcl of M-PER protein extraction reagent (Thermo, Waltham, MA) per approximately 20X106 washed cells. The concentration of proteins in lysates was determined with the colorimetric Pierce 660 nm protein assay (Thermo). For electrophoresis, lysates (40\u201360 \u03bcg of protein) were boiled for 5 min in buffer containing 50 mM Tris, 2% SDS and 143 mm \u03b2-mercaptoethanol. After electrophoresis in Mini-PROTEAN\u2122 TGX pre-cast 4%\u201315% gradient polyacrylamide gels (Bio-Rad, Hercules, CA) at 15\u201330 mA for 90 min, proteins were transferred overnight at 30 mA in 10% methanol-containing Tris-glycine buffer to 0.45 \u00b5 polyvinylidene difluoride membrane (GE Healthcare, Little Chalfont, UK). For immunoblotting, membranes were blocked in Tris-buffered saline (TBS; 10 mM Tris-HCl and 150 mM NaCl at pH 7.4) with 0.05% Tween-20 and 5% non-fat milk (Carnation\u2122, Nestle, Vevey, Switzerland), and then incubated in the same solution with a primary mouse monoclonal anti-SDHB antibody (ab14714, Abcam, Cambridge, MA) at room temperature for an hour. After washing in TBS/Tween-20 for 30 minutes, membranes were incubated in TBS/Tween-20/milk with a horseradish peroxidase-conjugated 10 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t secondary antibody for 1 hour at room temperature. Membranes were then washed in TBS/Tween-20 for 30 min, incubated in Luminata Forte\u2122 chemiluminescence reagent (Thermo), and exposed to photographic film (Thermo). Mouse anti-beta-actin (clone C4, Santa Cruz Biotechnology, Dallas, TX) and horseradish peroxidase-conjugated goat anti-mouse IgG (Invitrogen) antibodies were used at 1:200 and 1:2500 dilutions, respectively. Densitometry of Western blot signals was performed using the gel analysis method of ImageJ (version 1.47) after scanning the photographic films in grayscale at 400 dpi on an Epson Perfection v700 scanner (Schneider,Rasband & Eliceiri 2012). SDHB signals were normalized to those of beta-actin. conflict of interest disclosure : The authors declare no conflict of interest. 28 547 548 549 550 551 552 553 554 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t bora e. baysal1,\u2020, kitty de jong1, biao liu2, jianmin wang2, santosh k. patnaik3, paul k. : wallace1, r. thomas taggart1 : Departments of Pathology and Laboratory Medicine1 Biostatistics and Bioinformatics2 and Thoracic Surgery3, Roswell Park Cancer Institute, Buffalo, NY 14263, USA \u2020To whom correspondence should be addressed: Bora E. Baysal, MD, PhD Department of Pathology, Roswell Park Cancer Institute Elm & Carlton Streets, Buffalo, NY 14263, USA Tel: 716-845-3204; Email:bora.baysal@roswellpark.org 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t microscopy and imaging : Standard tissue culture monitoring and imaging was performed using an Axio microscope (Zeiss, Jena, Germany). Adherent aggregates (approximately 100 micron or larger in diameter) were counted at low power (2.5X) magnification. Live image photographs were taken using a Leica AF6000LX system (Houston, TX) which is comprised of a Leica DMI-6000B microscope and Leica LAS AF software interface. Nucleic acid isolation and analysis RNA and DNA were isolated using Trizol (Life Technologies, Grand Island, NY) and DNA Wizard genomic DNA purification kit (Promega, Madison, WI), respectively. Nucleic acid lysis buffer or Trizol was added directly to the adherent cells in plate well. Nucleic acid was quantified by a spectrophotometer (Nanondrop, Thermo Scientific, Wilmington, DE). Oligonucleotide primers and templates were obtained from IDT Technologies (Coralville, IA). 5 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t RT and qPCR were performed following the manufacturer\u2019s kits and instructions (LightCycler 480II, Roche, Indianapolis, IN). Complementary DNA from total RNA (1-2 \u00b5g) was synthesized using a mixture of random hexanucleotide and oligo-dT12 primer mixture. qPCR amplification of cDNAs was performed and crossing point (Cp) levels of C136U (T assay) and total SDHB (total assay) were determined separately. Each assay was performed in duplicate or triplicate wells. T assay and total assay had the same forward oligonucleotide primer for SDHB exon 1 and different oligonucleotide reverse primers in exon 2, where the C136U editing occurs. The reverse primer for the T assay had an extra T at its 3\u2019-end relative to the reverse primer in the common assay. (Oligonucleotide primers and probes used in this study are listed in supplemental Table S1.) The relative amount of the edited transcripts was calculated by the delta-Cp method by exponentiating the total transcript Cp minus the edited transcript Cp value to the power of 2. This approach assumes 100% duplication efficiency per cycle in both assays. Control PCR reactions were performed to test the specificity and amplification efficiency of oligonucleotide primer pairs using synthetic oligonucleotide templates. Assays with 8-fold serially diluted synthetic oligonucleotide templates for the wild-type and C136T edited sequences showed a duplication efficiency of 107% for both total and T assays between 1 femtomole and 0.245 attomole (Supplemental Table S2). The mutation-specific primer had a high specificity for the edited transcripts, with an average false positive amplification rate of 1.38% when the control wild type oligonucleotide template sequence was amplified in an over 25,000-fold concentration range. When 100% T template was used, the average estimate of the C136U edited transcripts was 91% between 8 femtomoles and 0.245 attomoles. These control experiments suggest that AS qPCR estimates may be slightly lower than the true editing rates. We chose this stringent AS qPCR method to ensure we did not overestimate the C136U editing rates. The estimated percentage of edited transcripts using this method was highly 6 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t reproducible in biological samples. For example, a set of 14 samples containing both low and high levels of edited transcripts gave very similar results in two replicate experiments with a Pearson correlation coefficient of 99.4%. In each assay, a positive control sample which had a mutation rate of 15%-17% was included. Relative quantification of cytidine deaminase (CDA) and SDHB mRNA expression was performed by 2^(-delta delta(Ct)) method (Livak & Schmittgen 2001) using expression levels of the housekeeping B2M beta-2 microglobulin transcripts for normalization. Microarray analysis of gene expression Expression profiling was accomplished using the Human HT-12 whole-genome gene expression array and direct hybridization assay (Illumina, San Diego, CA). Initially, 500 ng total RNA was converted to cDNA, followed by in vitro transcription to generate biotin-labeled cRNA using the Ambion Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX) as per manufacturer\u2019s instructions. 750 ng of the labeled probes were then mixed with hybridization reagents and hybridized overnight at 58 \u00b0C to the HT-12v4 BeadChips. Following washing and staining with Cy3-streptavidin conjugate, the BeadChips were imaged using the Illumina iScan Reader to measure fluorescence intensity at each probe. The intensity of the signal corresponds to the quantity of the respective mRNA in the original sample. The background corrected gene expression levels were extracted from BeadChip using Illumina\u2019s Genome Studio (v2011.1) gene expression module (v1.9.0). The log2-transformed expression levels were quantile normalized using lumi Bioconductor package in R (Du,Kibbe & Lin 2008) For data quality control, we excluded the genes with detection P value greater than 0.05 (i.e., indistinguishable from the background noise). 18941 out of 34686 genes passed this filtering for downstream analysis. 7 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t The limma Bioconductor package (Smyth 2004) was used to calculate the level of differential gene expression. Briefly, a linear model was fit to the data (paired design, with cell means corresponding to the different condition and a random effect for array) and selected contrasts of condition (i.e., case vs. control) were performed. A list of differentially expressed genes with P <0.05 and \u22652 fold-change was obtained and analyzed for enriched Gene Ontology (GO) categories and KEGG pathways using NCBI DAVID Bioinformatics Resources (Huang da,Sherman & Lempicki 2009) . The enriched GO terms and KEGG pathways with P <0.05 and \u22655 genes were kept. accession number : Microarray data have been deposited in the NCBI Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/) under accession number GSE45900. High-throughput RT-PCR amplicon sequencing SDHB and SDHD gene transcripts were amplified by RT-PCR with oligonucleotide primers targeting 5\u2019- and 3\u2019-untranslated regions (primer sequences can be obtained from authors) using Transcriptor reverse transcriptase (Roche) and Ultra2 Pfu high fidelity DNA polymerase (Agilent, Santa Clara, CA) . Libraries were preparedusing the Nextera XT DNA sample preparation kit (Illumina) for hHigh-throughput sequencing was on a MiSeq personal sequencing platform (Illumina). flow cytometry : Cells (0.5-1x106) were pelleted by centrifugation at 400g for 5 minutes and incubated for 20 minutes at room temperature in a total volume of 100 \u00b5l with a titrated cocktail of allophycocyanin (APC)-conjugated mouse anti-CD14 antibody (Invitrogen, Carlsbad, CA), Brilliant-violet 421 conjugated CD16 (BioLegend, San Diego, CA), phycoerythrin (PE)-conjugated mouse anti-CD163 antibody (Trillium Diagnostics, Brewer, ME), fluorescein isothiocyanate (FITC)-conjugated mouse 8 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t anti-CD206 (BD Biosciences, San Jose, CA), and PE-Cy7-conjugated mouse anti-HLA-DR antibody (BD Biosciences), followed by a wash in phosphate buffered saline (PBS; pH 7.4) and resuspension in 500 \u00b5l PBS. Viability is assessed by Live/Dead Yellow (Invitrogen). Flow cytometric acquisition was performed on a FACS Aria I flow cytometer (BD Biosciences) equipped with four laser excitation sources (405 nm at 30 mW; 488 nm at 15 mW; 561 nm at 30 mW, and 631 nm at 17 mW) that was quality-controlled on a daily basis by using BD cytometer setup and tracking beads and FACS DiVa software (BD Biosciences). The filter configurations for the PMTs measuring fluorescence emission of the applied fluorochromes were 450/50 nm (Brilliant-violet 421); 530/30 nm (FITC); 582/15 nm (PE); 780/60 nm (PE-Cy7); and 660/20 nm (APC). Autofluorescence and single-color controls were acquired to perform spectral overlap compensation using the automated compensation matrix feature in FACS DiVa software. A forward scatter threshold was applied to eliminate electronic noise and small particles from the flow cytometric acquisition. A total of 20,000 scatter-inclusive events were acquired for each specimen. Data analysis was performed with FlowJo software version 10.0.4 (Tree Star, Ashland, OR). isolation of cd14+ monocytes : Monocytes were isolated either by flow cytometric gating of CD14+ cells (both dim and strong intensity) on a BD FACS Aria I flow sorter, or by CD14 microbeads (Miltenyi Biotec Inc. Auburn, CA) following manufacturer\u2019s protocol. Flow cytometry showed 93% or higher purity after CD14 purification. immunocytochemical staining : PBMCs were grown in tissue culture chamber slides (Labtek, Scotts Valley, CA). Before staining, medium was aspirated and adherent cells were washed once with PBS. Adherent cells were fixed by 9 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t alcohol-based cytology fixative (Leica) and air dried. For antigen retrieval, slides were heated in the steamer for 20 minutes in a citrate buffer (pH 6.0), followed by a 20 minute cool down. Endogenous peroxidase was quenched with aqueous 3% H2O2 for 10 minutes and washed with PBS with 0.05% Tween 20 (Sigma). Slides were loaded on anautostainer (Dako, Carpinteria, CA) and a serum-free protein blocker (catalog #X0909, Dako) was applied for 5 minutes, blown off, and the antibody applied for one-hour. Biotinylated goat anti-mouse IgG (Jackson Immuno Research Labs, catalog #115-065-062) was applied for 30 minutes, followed by the Vectastatin Elite ABC Kit (Vector Labs, Burlingame, CA) for 30 minutes, and the 3, 3'-diaminobenzidene chromagen (Dako) for 5 minutes. Finally, the slides were counterstained with hematoxylin, dehydrated, cleared and cover slipped. c136u editing rate increases during short-term culture of pbmcs in a time-dependent : manner We cultured monocyte-enriched PBMCs for in vitro macrophage differentiation by plate adherence in the presence of 10% fetal bovine serum (FBS) at standard culture conditions as described (Roiniotis, et al., 2009). Flow cytometry showed that the fraction of CD14+ cells among the adherent cells on culture days 5-6 (25.9% \u00b1 5.2%; n=4) was comparable to the uncultured monocyte-enriched PBMC samples (27.2%\u00b1 2.7%; n=10). Initial cultures showed that the fraction of the edited transcripts increased in the adherent cells on days 5-7 then decreased to baseline levels on later days (Supplemental Figure S1A). Short-term cultures showed that the editing rates were lower in the first three days than on days 5-7 (3.54% versus 11.62%, n=15, P=8X10-4, Wilcoxon matched pairs signed ranks test, Figure 1C). The uncultured monocyte-enriched PBMC samples had lower C136U editing rates than their matched cultures on days 5 and 6 in 17 of the 17 samples (1.7% \u00b1 0.2% versus 10.06% \u00b1 0.8%; P=2.9X10-4, Wilcoxon matched pairs signed ranks test). To confirm monocyte origin of the edited transcripts in the cultured samples, we performed flow cytometric sorting of the attached CD14+ cells from culture days 5-8 which essentially separated monocyte/macrophages from lymphocytes to >95% purity. In five of five samples, the CD14+ cells showed higher editing rates than the CD14- cells (Figure 1D). The editing rate was higher among the adherent cells than those in the supernatant (Figure 1E). Collectively, these findings indicate that C136U editing is induced in monocyte/macrophage lineage cells during culture of monocyte-enriched PBMCs in a time-dependent manner. 14 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t The maximum mutation rate reached on days 4-8 varied markedly among samples from 1.2% to 18.8%. Total number of cultured cells per well correlated positively but very weakly with the maximum mutation rate (Supplemental Figure S1B). The maximum C136U editing rate trended higher when the total number of monocyte-enriched PBMCs was over 21 million per well versus 20 million or less per well, though this difference did not reach statistical significance (10.9% \u00b1 1.23%; n=16 versus 8.54% \u00b1 1.03%; n=15; P=0.1; Mann Whitney U test, one-tailed). Morphologic evaluation of cultures associated with C136U editing We noted the development of three dimensional adherent aggregates (AAs) in monocyte-enriched PBMC cultures, especially in those that had the highest C136U editing rates. These aggregates were detectable within 12 hours and increased in density and size up to a week in culture (Figure 2A and 2B). Hourly photographic imaging in the first two days showed that the aggregates start as a loose collection of cells and become larger and more compact by merging and recruitment of individual cells (Supplemental video S1). Cultures with low AA densities showed lower mutation rates than those with intermediate or high AA densities (n=16, P=0.015, Mann-Whitney U test; Figure 2C). Morphologic examination showed that the AAs were primarily comprised of intermediate-size cells that generally had round non-convoluted nuclei and a moderate amount of cytoplasm. Occasional lymphocytes and large monocytoid cells with curved nuclei were also noted within the aggregates. In contrast, numerous individually attached macrophages, including those with spindle-like morphology or multi-nucleation, had more abundant cytoplasm compared to cells in the AAs (Figure 2D). Immunostaining with an anti-CD163 antibody confirmed that most cells in the AAs were aggregated monocytes/macrophages (Figure 2E). These results suggest that C136U editing primarily occurs in aggregating monocytes that are in the process of differentiating to macrophages. 15 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t Microarray analysis of differentially expressed genes associated with increased C136U editing in normoxia We performed pairwise microarray gene expression analysis of four sets of low-editing (day 3) and high-editing (day 5-8) samples cultured in normoxia (Table 1) to evaluate (a) global changes in cellular pathways; (b) whether adherent aggregates are associated with micro-environmental hypoxia; and (c) changes in the SDH subunit and cytidine deaminase family of genes. Analysis showed that 171 genes were upregulated and 123 genes were downregulated genes by at least two-fold at a statistically significant level (P <0.05). The subset of these genes (n=55) that showed \u2265 fold-change is shown in Supplemental Table S3. The complete list is available on GEO database under accession number GSE45900. No SDH subunit or APOBEC family gene met these criteria. Cytidine deaminase (CDA) was the only gene from the cytidine deaminase family of genes that had its expression level changed significantly, with a 3.1-fold increase. qPCR analysis confirmed upregulation of CDA expression (average 3.7-fold) and little change in SDHB expression (average 0.98-fold) in the high-editing samples relative to the low-editing ones. Significant gene expression changes coordinately occurred in functionally related genes in several Gene Ontology (GO) categories (n=159). This number decreased to 10 after Bonferroni correction for multiple testing was applied (Supplemental Table S4). These 10 categories included defense to wounding, inflammatory, immune and defense responses, taxis, chemotaxis, carboxylic and organic acid transport, locomotory behavior and regulation of cell proliferation. Notably, hypoxia-response genes were not categorically different between the low and the high editing samples. These results indicate that significant gene expression changes occur in inflammatory and immune response pathways during increase in SDHB editing and that CDA is a candidate gene for enzymatic C136U deamination. 16 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t Flow cytometric characterization of cultures associated with C136U editing To evaluate monocyte macrophage maturation in PBMC culture, we performed flow cytometric characterization of the attached cells using monocyte/macrophage-associated antibodies for CD14, CD16, CD163, HLA-DR and CD206, a mannose receptor associated with M2 type macrophage polarization that promotes wound healing (Porcheray, et al., 2005). Cold-aggregated uncultured PBMCs showed a discrete and relatively uniform monocytic population that is positive for CD14, HLA-DR and CD163; but negative for CD206 and largely negative for CD16 (Figure 3). Cultured adherent cells at day 6, which showed an increased editing rate, contained a CD14+ population comprised of larger cells with more complexity and were dim-positive for CD206, heterogeneously positive for CD14 and brightly positive for HLA-DR. CD16 and CD163 patterns were similar and showed a heterogeneous negative-to-positive range. Analysis of the adherent cells at day 15, when the editing rates are typically low and the AAs are loose (Supplemental Figure S1C), showed a mature macrophage population that had increased complexity and a more uniform antigenic profile, including homogenous positivity for CD14, CD16, CD163, HLA-DR and CD206. Taken together, these results demonstrate that high C136U editing peaks during monocyte macrophage differentiation in normoxic culture. When monocyte-enriched PBMC cultures were treated with M-CSF and GM-CSF/IL4 to facilitate macrophage and dendritic cell differentiation, respectively, lower editing rates were seen compared to the control cultures that contained only 10% FBS (P=0.012, n=8, and P=0.04, n=5, respectively, Wilcoxon matched pairs signed ranks test Supplemental Figure S1D). We also tested C136U editing 17 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t rates in the monocytic leukemia cell line THP-1. We added macrophage differentiation agents to the media with 10% FBS including phorbol ester PMA, retinoic acid and vitamin D. Significant C136U editing was not identified (less than 1%) either in the untreated THP-1 cell line or after treatment with the differentiating agents. Similarly, hypoxia exposure of the THP-1 cell line showed no increase in C136U levels on days 1, 2 and 3 (less than 1%). These results indicate that C136U editing primarily occurs in mature monocytes during differentiation to macrophages in the presence of serum under normoxic culture conditions and that cytokine-driven macrophage or dendritic cell differentiation or leukemic transformation reduces editing rates. Induction of C136U editing by hypoxia Because tissues with inflammation and infarction harboring monocyte/macrophage lineage cells are often hypoxic and because monocyte-macrophage differentiation occurs in tissues where oxygen pressure is lower than that in blood (Roiniotis, et al., 2009), we evaluated the role of hypoxia on C136U editing. We observed that monocyte-enriched PBMC cultures in hypoxia did not develop the firmly adherent aggregates associated with high editing rates in the normoxic cultures and that essentially all cells were in suspension. The fraction of viable CD14+ monocytes in the hypoxic supernatants on culture day 2 was similar to the original uncultured samples as evaluated by flow cytometry (20.9% versus 20.4%, n=4). The editing rate in the hypoxic cultures increased in the first three days with a peak on day 2, when it is typically low in the normoxic control cultures (Figure 1C and 4A). In five of the 14 samples, C136U editing rates were higher (21%-49%) than in any normoxic culture. Hypoxia increased the fraction of C136U transcripts by 0.4% to 46% on culture day 2. Pairwise comparison of the editing rates in supernatant cells in hypoxia versus total cells in normoxia from 14 different samples in the first three days showed marked upregulation of C136U editing by hypoxia (P=2X10-7, n=42, Wilcoxon matched pairs test; Fig. 4B). Hypoxia increased 18 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t C136U fractions in 39 of 42 pairwise comparisons from 14 samples cultured in normoxia and hypoxia for three days. Comparison of the flow sorted CD14+ and CD14\u2013 cell populations confirmed that monocytes were the main source of C136U editing in hypoxia (Figure 4E). RT-qPCR analysis showed marked upregulation of CDA (average 7.5-fold change on day 2, n=6) and downregulation of SDHB (average 0.37-fold change on day 3, n=6) in the hypoxic cultures relative to the control gene beta 2 microglobulin. Western blot analysis of a monocyte-enriched PBMC sample cultured in normoxic and hypoxic conditions showed no marked changes in the expression SDHB protein product (Figure 4D), suggesting that the impact of C136U editing on protein expression levels is subtle and quantitative. The variable increase in the percentage of C136U transcripts by hypoxia could be caused by unknown technical or biological factors including variable fraction of monocytes in donors, variation in inherent enzymatic activity of the putative editing enzyme or nonsense mediated decay pathway activation.(Chang, Imam & Wilkinson 2007). Analysis of genomic DNA for C136T editing To test whether the C136U RNA mutation has a corresponding C-to-T genomic DNA mutation in SDHB exon 2, we compared mutant RNA and DNA levels using our previously described semi-quantitative method based on PCR amplification and Taq1 restriction enzyme digestion (Baysal 2007). We found no evidence of a corresponding DNA mutation in the hypoxic samples containing high levels of C136U RNA mutations (Figure 4C). These results indicate that C136U mRNA editing occurs during or after transcription. High throughput sequencing of SDHB and SDHD transcripts To obtain a global view of transcript editing in the SDHB and the SDHD genes, we performed high throughput sequencing of the full length coding transcripts obtained by RT and high-fidelity PCR. 19 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t We sequenced the four pairs of normoxic cultures that were characterized by microarray gene expression analyses. Each pair derived from the same PBMC and contained a sample with a low C136U editing rate (day 3 culture) and a sample with a high C136U editing rate (day 5-8 culture). Interrogation of the transcript sequences confirmed C136U mutations in each of the high-editing-rate sample. On average, 40% higher editing levels were seen by high throughput sequencing than those estimated by AS qPCR (Table 1). No additional canonical RNA edits in the form of A-to-I/G or C-to-U/T changes were identified in the SDHB transcripts. No additional SDHB transcript variants could be identified by Sanger sequencing. In contrast, Sanger sequencing confirmed C136U in all four samples that showed high editing rates by AS qPCR and by high-throughput sequencing (Supplemental Figure S1E). Similarly, SDHD transcripts showed no evidence of RNA editing by high-throughput sequencing. Taken together, these results demonstrate that SDHB C136U editing is not accompanied by other transcript variants either in SDHB or SDHD and suggest that a site-specific C-to-U RNA editing mechanism regulates the SDHB functional transcript dosage. Analysis of transcriptome sequence databases We examined RNA sequencing data of the Illumina Human Body Map and ENCODE Human Transcriptome projects (Derrien, et al., 2012; Tilgner, et al., 2012; Brazma, et al., 2003) for sequence variations in the ORF region SDHB mRNA. As expected, among the 16 normal human tissues from Human Body Map, the highest levels of SDHB C136U editing was seen in white blood cells (1.7%), with the other 15 tissues showing an average editing level of 0.2% (SD=0.3%), with ovary (1%) and kidney (0.9%) being those with the highest levels among the 15 (Supplemental Table S5). Among the 45 samples of 23 different cell-types of the ENCODE set, the editing rate was highest for the two primary CD14+ monocytes (1.9% and 2.6%), with the other samples showing an average 0.1% (SD=0.2%) editing rate (Supplemental Table S6). The editing rates noted for the samples for white 20 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t blood cells and the monocytes are unlikely to be a result of sequencing error since a base call of U instead of C was made in only 0.04%-0.09% of the reads when all 213 C-bearing positions of the SDHB coding region were examined (P <0.001, Chi square test). These results support our earlier conclusions that C136U editing occurs at low but statistically significant levels in peripheral blood monocytes. authorship : BEB conceptualized the study and wrote the manuscript. BEB, RT, JW and PKW designed experiments. RT, KDJ and BEB performed experiments. BL and JW performed the bioinformatic analysis of microarray expression data. SP performed the ENCODE and Illumina database analysis. All authors approved the final manuscript. 27 542 543 544 545 546 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t supplemental material : Supplemental Figure S1 (A) Impact of long-term culture and cell density on C136U editing are shown. Five monocyte-enriched PBMCs were cultured long-term and the C136U editing rates were measured. The editing rate was higher in adherent cells on days 5-7 than in the uncultured monocyte-enriched PBMCs and on days 12-14 and 19-21 (P=0.017, n=5, one-way ANOVA). (B) Maximum C136U editing rate in the adherent cells shows very weakly positive correlation with the total number of monocyte-enriched PBMCs per well. Total number of samples=31. (C) Attached aggregates (AAs) on culture day 20 are shown. The AAs start loosening after one week in culture and eventually appear as flat areas of increased cellular density. This culture was fed with fresh RPMI-1640/10% FBS on day 8. (D) Cytokine mediated differentiation towards macrophages or dendritic cells reduce C136U editing rates. Cultures containing 10% FCS showed higher mutation rates than those treated with M-CSF (p=0.012) or GM-CSF/IL4 (p=0.04). Multi-day averages from two PBMC cultures are shown. Matched samples were collected on days 4-8 for the m-CSF culture (n=8) and days 4-6 for the GM-CSF/IL4 cultures (n=5). The editing rate was obtained from adherent cells in 10% FCS and m-CSF treated (macrophage-differentiation) wells and from non-adherent cells in supernatant in GM-CSF/IL4-treated (dendritic cell-differentiation) wells. (E) Sanger sequencing confirms C136U RNA editing in all four samples that showed high editing rates by RT-qPCR and high-throughput sequencing. Chromatograms show examples of a sample with a low editing rate (sample 1, day3 in Table 1) and one with a high editing rate (sample 4, day 7 in Table 1). 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Human Pathology 4: 251. 34 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t Sanjana NE, Levanon EY, Hueske EA, Ambrose JM, Li JB. 2012. Activity-dependent A-to-I RNA editing in rat cortical neurons. Genetics 192: 281. Schneider CA, Rasband WS, Eliceiri KW. 2012. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9: 671. selak ma, armour sm, mackenzie ed, boulahbel h, watson dg, mansfield kd, pan y, : Simon MC, Thompson CB, Gottlieb E. 2005. Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase. Cancer Cell 7: 77. Skuse GR, Cappione AJ, Sowden M, Metheny LJ, Smith HC. 1996. The neurofibromatosis type I messenger RNA undergoes base-modification RNA editing. Nucleic Acids Research 24: 478. Smyth GK. 2004. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology 3: Article3. tilgner h, knowles dg, johnson r, davis ca, chakrabortty s, djebali s, curado j, : Snyder M, Gingeras TR, Guigo R. 2012. Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs. Genome Research 22: 1616. Uitenbroek DG SISA Binomial. 2013. http://www.quantitativeskills.com/sisa/distributions/binomial.htm. Last accessed 4/4/2013. 35 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t Table 1(on next page) Percentage of C136U transcripts estimated by allele specific (AS) qPCR and high-throughput amplicon sequencing in normoxic cultures PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t Table 1 Percentage of C136U transcripts estimated by allele specific (AS) qPCR and high-throughput amplicon sequencing in normoxic cultures Sample No. Day in culture AS qPCR high-throughput sequencinga 1 Day 3 2.2% NRb 1 Day 6 15.9% 22% 2 Day 3 1.3% NR 2 Day 8 12.3% 23% 3 Day 3 4% NR 3 Day 6 13.5% 15% 4 Day 3 8.7% 11% 4 Day 7 18.8% 26% a Sequencing depth ranges between 4980 and 4986 for the reported variants. b NR= Not reported. Variants detected by high-throughput sequencing are reported only when their frequency exceeds 10%. PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t Figure 1 SDHB C136U RNA editing is induced in monocytes during short-term culture. (A) C136U editing is measured in 9 lymphoblastoid cell lines and one fibroblastic cell line, freshly isolated PBMCs (n=50), PBMCs monocyte-enriched by cold-aggregation (Mono+; n=42) and monocyte-depleted PBMCs comprised of largely lymphocytes (Mono-; n=10). Horizontal lines represent mean \u00b1 standard errors throughout the figures. (B) Fraction of the C136U transcripts weakly correlates with the CD14+ monocyte percentage in monocyte-enriched PBMCs (Pearson correlation coefficient r=0.36). (C) Impact of short-term culture on C136U editing. Short-term culture of five monocyte-enriched PBMCs shows upregulation of C136U editing in adherent cells in culture days 5-7 compared to days 1-3. Day 0 represents the uncultured samples. (D) Flow cytometric sorting of CD14+ versus CD14- adherent cells on culture days 5-8 shows higher mutation rates in monocyte-macrophage lineage than in lymphocytes (P=0.04, n=5, Wilcoxon matched pairs signed ranks test). (E) C136U editing is lower among non-adherent cells in the supernatant than in adherent cells (4.3% versus 9.8%) on culture days 5-7 (n=19 wells from 11 samples on days 5-7, P=2.1 X10 -4 , Wilcoxon matched pairs signed ranks test). PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t Figure 2 SDHB C136U RNA editing rate correlates with adherent monocyte-rich adherent aggregates (AA) in normoxic culture. The AAs on culture days 1 (A) and 5 (B) are shown (5X low power field; vertical bars equal to 1 mm). The culture is initiated with 30 million cells per well. The AAs grow in size until approximately days 5-7 when the C136U editing rate also usually peaks. (C) High editing rates are seen in cultures with intermediate and high density of adherent aggregates. Aggregate densities are grouped as follows: Low= less than 10 aggregates/per 5X low power field (lpf); Intermediate=10-20 aggregates/lpf; High= more than 20 aggregates/lpf (D) Giemsa stain highlights an adherent aggregate on day 6 culture. Individually attached mature macrophages have mostly round eccentric nuclei and abundant cytoplasm. Occasional multi-nucleated macrophages (arrowheads) and rare small lymphocytes (green arrows) are also present. Macrophages within the aggregate are smaller with less cytoplasm and occasionally have curved nuclei (arrows). (E) CD163 immunostaining of adherent aggregates. Immunocytochemical staining for monocyte-macrophage-specific antigen CD163, a scavenger receptor for hemoglobin-haptoglobin complex, demonstrates that the adherent aggregates are primarily comprised of monocyte/macrophage lineage cells. Individually attached macrophages outside the aggregate also stain with variable intensity (short arrows); while numerous small lymphocytes are negative (long arrows). PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t Figure 3 SDHB C136U RNA editing increases during monocyte-macrophage maturation. Flow cytometric evaluation of monocyte-macrophage maturation is shown in uncultured cold aggregated PBMCs (first column), adherent cells on culture day 6 (second column) and adherent cells on culture day 15 (third column). The day 15 culture shows a relatively homogenous large (indicated by high forward scatter, FSC-A) macrophage population that has high complexity (indicated by high side scatter, SSC-A) and is positive for CD206 (mannose receptor), HLA-DR and CD163. In contrast, uncultured monocytes are largely a uniform population that is smaller with less complexity and is negative for CD206. The day 6 culture, which has 11.6% C136U editing rate, shows a CD14+ population that is brightly positive for HLA-DR, dim-positive for CD206 and negative for CD163. CD14+ monocyte-macrophage and lymphocyte populations are marked by green and red, respectively. PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t Figure 4 Hypoxia induces SDHB C136U RNA editing. (A) The editing rates are higher on days 1-3 in hypoxia and on days 5, 6 in normoxia. The average of four independent cultures is presented. (B) C136U editing rates in 12 independent cultures in hypoxia versus normoxia during 3 days of culture are shown. The editing rates were obtained from supernatant in all hypoxic cultures (first column) and from total cell population in normoxic cultures, except in two normoxic cultures where only supernatant was collected (second column). D0 samples represent the original uncultured cold aggregated PBMCs. (C) PCR amplification and Taq1 restriction enzyme (RE) digestion shows no evidence of C136T DNA mutation in SDHB exon 2 genomic DNA (gDNA) (right panel) in the same samples with high C136U RNA editing rates (left panel). The C13U RNA editing rates above the lanes were measured by AS qPCR. RT- and gDNA-PCRs generate amplicon sizes of 285 bp and 233 bp, respectively. Taq1 RE digestion of wild type cDNA and gDNA sequences generates 159 bp/126 bp and 131 bp/102 bp bands, respectively. C136U/T mutation destroys the RE site. (D) Western blot analysis of normoxic (N) and hypoxic (H) cultures from one donor shows no major changes in SDHB protein expression, normalized against beta actin, as the editing rate increases in hypoxia, but possibly a subtle decrease when the C136U percentage is 16.1% on day 2 in hypoxia. Hypoxic cultures were sampled twice a day, 10 h apart. (E) The editing rates are shown for flow sorted CD14+ and CD14- viable cells from day 2 hypoxic cultures (n=4). In all experiments, hypoxic and control normoxic culture derive from the same donor (i.e., oxygen tension is the only variable). PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:588:0:0:NEW 13 Jun 2013) R ev ie w in g M an us cr ip t",
    "url": "https://peerj.com/articles/153/reviews/",
    "review_1": "Maria Deli \u00b7 Aug 15, 2013 \u00b7 Academic Editor\nACCEPT\nThe revision addressed all the criticism raised by the reviewers.",
    "review_2": "Maria Deli \u00b7 Jun 17, 2013 \u00b7 Academic Editor\nMAJOR REVISIONS\nSome major technical issues (PCR, western) were raised by the reviewers. Functional significance of the observed changes need to be proven. Additional experiemnts are required.",
    "review_3": "Reviewer 1 \u00b7 Jun 15, 2013\nBasic reporting\n1. The structure of the \"Introduction\" should be rearranged in order to stress cannabidiol (CBD), the molecul of the scope, as promissing candidate for clinical utilization and thus it is of high importance to know whether CBD influences the expressionof P-gp and/or BCRP.\n2. Authors should explain in introductory part if there is chance to use CBD therapeutically in pregnancy.\n3. Page 2, line 13, word polarizer shoud be changed to polarized\n4. Source of cells should be given more precisely, and the studies, in which the cells were used should be refered, since various clones of MCF-7 and BeWo cells exist differing in P-gp and/or BCRP expression (e.g. Ceckova et al. 2006)\n4. Page 4, line 12, citations Golan et al 2009 and Feinshtein et al 2010 are provided to describe cell cultivation. These citation should be reconsider as only the latter deals with JAr cell lines. MCF-7 and Bewo cultivation is not described.\n5. page 5, line 25, name of the company producing High capacity cDNA RT kit should be provided\n6. Word chronic when speaking about exposure should be omitted from the text of the manuscript\nExperimental design\n1. PCR analysis should be remade, using one housekeeping gene for the analysis is not sufficient to achieve desired accuracy, especially in this study, where differences in mRNA expresssion are 2-2,5 fold at maximum.\n2. Results desribing expression of P-gp/BCRP in all cell lines used is incosistent. Immunocytochemistry of BCRP should be added (figure 1)\n3. Description of the results from protein level to mRNA is questionable. Authors should reconsider this scheme and to think about semi-quantitative analysis of protein expression usin immunohistochemistry instead of qRT-PCR as mRNA analysis seems as in this context as a step back\nValidity of the findings\n1. Figure 2 - the results provided seem to be questionable. Based on the analysis of actin or Na+/K+ ATPase it is obvious that protein load in gel was not the same everywhere, moreover, intensity of bands of these proteins correlates with intensity of bands of target proteins, indicating that the changes in protein expression observed are not correct. Does the software used EZQuant-Gel 2.11 normalize the target protein amount to reference protein properly? Moreover, in e.g. figure A for 72 h, it seems unlikely that 1.25 fold increase with SD +- 20% has P equal to **, especially when one-way ANOVA is used for three replicates. So, how many biological replicates were used as source data for analysis, it should be stated precisely. Similarly in figure B, decrease in BCRP protein expression for 72 h in BeWo should be discussed\n2. Figure 3 (B,D), the wester blots as provided cannot be used for semi-quantitative analysis, the results as shown seem to be irrelevant.\n3. Figure 4, picture of western blot is completely lacking\n4. Figure 5, again authors must state how many samples (it means biological replicates) have been used for the analysis and the same number of samples must be used for all calculations. This figure shows the higher SD the better statistical significancy despite the same increase (2 fold), moreover, the changes observed can be the result of instability of the housekeeping used. This must be corrected. Comments concerning figure 2-4 raise dobts about validity of the study outcomes.\n5. P-gp and BCRP are considered to have similar transcriptional regulation, authors should discuss the opposing effect of CBD on P-gp and BCRP.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines (v0.1)\". PeerJ https://doi.org/10.7287/peerj.153v0.1/reviews/1",
    "review_4": "Reviewer 2 \u00b7 Jun 11, 2013\nBasic reporting\nThis manuscript is clearly written and conforms to professional standards. The background information provided in the introduction and discussion is excellent. The structure of the article conforms the journal style, and the figures are nicely composed and labeled.\nExperimental design\nThe research question is clearly stated, the experiments are well designed, the methods are well described, and the investigations have been conducted rigorously.\nValidity of the findings\nThe data is strong and is presented in a clear and well understandable way. The results make sense in the context of the research hypothesis. The conclusions are appropriate for the findings.\nAdditional comments\nThis is an excellent study which aimed to investigate the effect of cannabidiol on the expression of ABC transporters P-gp and BCRP in trophoblast-like cells. This is important since cannabis use is an increasing problem during pregnancy, and its potential consequences on the placenta and the fetus are of major concern. The manuscript is insightful and very well written, the statistics and the representation of the data are proper, and the paper is easily readable. Therefore, I suggest to have it accepted for publication, and I have only minor comments to be addressed before publication:\n\n1) In several places all along the text some periods are misplaced. This needs to be fixed.\n2) Page 2: Please write \u201cpolarized\u201d instead of \u201cpolarizer\u201d.\n3) Please rephrase the sentence on Page 2, line 12, since the word \u201cconstructed\u201d does not seem to be the best fit.\n4) Page 2, line 20: Please write \u201cprotein\u201d after kDa.\n5) I would write \u201ctrophoblast-like\u201d instead of \u201ctrophoblast\u201d cell lines, since JAR and BeWo cells are choriocarcinoma cell lines.\n6) On Page 4, I would put \u201ch\u201d: after 24 and 48.\n7) On Page 5: Please describe the antibodies used for the Na/K ATPase and NFkB Western blots.\n8) DAPI (4',6-diamidino-2-phenylindole) is written with capital letters. Please define first and write this way.\n9) Please provide vendor information for DAPI.\n10) On Page 6: correctly it is called Delta-Delta Ct method.\n11) Part of the statistical analysis is missing, which needs to be added.\n12) Page 8, lines 1 and 110: The use of \u201cplacenta\u201d is not correct in this context.\n13) It would be useful if subheadings could be used in the Discussion.\n14) Reference is missing from the sentence ending on Page 10/line 13,\n15) Figure legends are uploaded twice.\n16) Figure 1: Please define \u201cNC\u201d.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines (v0.1)\". PeerJ https://doi.org/10.7287/peerj.153v0.1/reviews/2",
    "pdf_1": "https://peerj.com/articles/153v0.2/submission",
    "pdf_2": "https://peerj.com/articles/153v0.1/submission",
    "review_5": "Reviewer 3 \u00b7 Jun 7, 2013\nBasic reporting\nNo comments\nExperimental design\nThe study is very preliminary and completely descriptive. I have several concerns regarding the experimental design and the interpretations of the results.\nValidity of the findings\nNo comments\nAdditional comments\nIn this study the authors have investigated the effect of cannabidiol on two cell lines derived from human choriocarcinoma. The conclusions are based on descriptive data of mRNA and protein abundance. Nevertheless the results are discussed as if the changes observed would affect the placental barrier.\n\nMajor points:\n\n1. Previous studies have already shown the expression in BeWo and JAr cell lines of a complete panel of transporters and pumps, in addition to MDR1 and BCRP (see for instance Serrano et al., Placenta 28 (2007) 107-117). Several results described here as original (for instance page 7, lines 6-8) should be commented as mere confirmation of previous reports.\n\n2. Page 8, line 9. The present paper offer little clue on the mechanism of action of cannabidiol on the expression of these two ABC proteins. The fact that mRNA levels were consistent with protein abundance is an interesting data but adds little to the mechanism controlling the expression of these genes.\n\n3. Are the observed effects due to specific or non-specific (for instance chemical stress-mediated) mechanisms? Are cannabinoid receptors expressed in JAr and BeWo cells? Are they responsive to cannabidiol? What is the intracellular signalling pathway involved? Is there any evidence for a role of any nuclear receptor?\n\n4. Page 11, lines 15-22. This paragraph is highly speculative. Are the authors proposing an autocrine mechanism? What are the meadiators?\n\n5. Page 12, lines 8-13. The conclusion is again very speculative, above all, the last sentence.\n\n6. Figure 1. Why BCRP was not included in this part of the study?\n\n7. Figure 1. Immunolocalization of MDR1 does not seem to be at the plasma membrane of BeWo and JAr cells, but intracellular. Is BCRP also located intracellularly? How this fit the results from the western blot analyses?\n\n8. Does the treatment with cannabidiol change the subcellular localization of these proteins?\n\n9. Any functional study would be required to suggest that cannabidiol is able to change the role of these proteins in the placental barrier.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #3 of \"Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines (v0.1)\". PeerJ https://doi.org/10.7287/peerj.153v0.1/reviews/3",
    "all_reviews": "Review 1: Maria Deli \u00b7 Aug 15, 2013 \u00b7 Academic Editor\nACCEPT\nThe revision addressed all the criticism raised by the reviewers.\nReview 2: Maria Deli \u00b7 Jun 17, 2013 \u00b7 Academic Editor\nMAJOR REVISIONS\nSome major technical issues (PCR, western) were raised by the reviewers. Functional significance of the observed changes need to be proven. Additional experiemnts are required.\nReview 3: Reviewer 1 \u00b7 Jun 15, 2013\nBasic reporting\n1. The structure of the \"Introduction\" should be rearranged in order to stress cannabidiol (CBD), the molecul of the scope, as promissing candidate for clinical utilization and thus it is of high importance to know whether CBD influences the expressionof P-gp and/or BCRP.\n2. Authors should explain in introductory part if there is chance to use CBD therapeutically in pregnancy.\n3. Page 2, line 13, word polarizer shoud be changed to polarized\n4. Source of cells should be given more precisely, and the studies, in which the cells were used should be refered, since various clones of MCF-7 and BeWo cells exist differing in P-gp and/or BCRP expression (e.g. Ceckova et al. 2006)\n4. Page 4, line 12, citations Golan et al 2009 and Feinshtein et al 2010 are provided to describe cell cultivation. These citation should be reconsider as only the latter deals with JAr cell lines. MCF-7 and Bewo cultivation is not described.\n5. page 5, line 25, name of the company producing High capacity cDNA RT kit should be provided\n6. Word chronic when speaking about exposure should be omitted from the text of the manuscript\nExperimental design\n1. PCR analysis should be remade, using one housekeeping gene for the analysis is not sufficient to achieve desired accuracy, especially in this study, where differences in mRNA expresssion are 2-2,5 fold at maximum.\n2. Results desribing expression of P-gp/BCRP in all cell lines used is incosistent. Immunocytochemistry of BCRP should be added (figure 1)\n3. Description of the results from protein level to mRNA is questionable. Authors should reconsider this scheme and to think about semi-quantitative analysis of protein expression usin immunohistochemistry instead of qRT-PCR as mRNA analysis seems as in this context as a step back\nValidity of the findings\n1. Figure 2 - the results provided seem to be questionable. Based on the analysis of actin or Na+/K+ ATPase it is obvious that protein load in gel was not the same everywhere, moreover, intensity of bands of these proteins correlates with intensity of bands of target proteins, indicating that the changes in protein expression observed are not correct. Does the software used EZQuant-Gel 2.11 normalize the target protein amount to reference protein properly? Moreover, in e.g. figure A for 72 h, it seems unlikely that 1.25 fold increase with SD +- 20% has P equal to **, especially when one-way ANOVA is used for three replicates. So, how many biological replicates were used as source data for analysis, it should be stated precisely. Similarly in figure B, decrease in BCRP protein expression for 72 h in BeWo should be discussed\n2. Figure 3 (B,D), the wester blots as provided cannot be used for semi-quantitative analysis, the results as shown seem to be irrelevant.\n3. Figure 4, picture of western blot is completely lacking\n4. Figure 5, again authors must state how many samples (it means biological replicates) have been used for the analysis and the same number of samples must be used for all calculations. This figure shows the higher SD the better statistical significancy despite the same increase (2 fold), moreover, the changes observed can be the result of instability of the housekeeping used. This must be corrected. Comments concerning figure 2-4 raise dobts about validity of the study outcomes.\n5. P-gp and BCRP are considered to have similar transcriptional regulation, authors should discuss the opposing effect of CBD on P-gp and BCRP.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines (v0.1)\". PeerJ https://doi.org/10.7287/peerj.153v0.1/reviews/1\nReview 4: Reviewer 2 \u00b7 Jun 11, 2013\nBasic reporting\nThis manuscript is clearly written and conforms to professional standards. The background information provided in the introduction and discussion is excellent. The structure of the article conforms the journal style, and the figures are nicely composed and labeled.\nExperimental design\nThe research question is clearly stated, the experiments are well designed, the methods are well described, and the investigations have been conducted rigorously.\nValidity of the findings\nThe data is strong and is presented in a clear and well understandable way. The results make sense in the context of the research hypothesis. The conclusions are appropriate for the findings.\nAdditional comments\nThis is an excellent study which aimed to investigate the effect of cannabidiol on the expression of ABC transporters P-gp and BCRP in trophoblast-like cells. This is important since cannabis use is an increasing problem during pregnancy, and its potential consequences on the placenta and the fetus are of major concern. The manuscript is insightful and very well written, the statistics and the representation of the data are proper, and the paper is easily readable. Therefore, I suggest to have it accepted for publication, and I have only minor comments to be addressed before publication:\n\n1) In several places all along the text some periods are misplaced. This needs to be fixed.\n2) Page 2: Please write \u201cpolarized\u201d instead of \u201cpolarizer\u201d.\n3) Please rephrase the sentence on Page 2, line 12, since the word \u201cconstructed\u201d does not seem to be the best fit.\n4) Page 2, line 20: Please write \u201cprotein\u201d after kDa.\n5) I would write \u201ctrophoblast-like\u201d instead of \u201ctrophoblast\u201d cell lines, since JAR and BeWo cells are choriocarcinoma cell lines.\n6) On Page 4, I would put \u201ch\u201d: after 24 and 48.\n7) On Page 5: Please describe the antibodies used for the Na/K ATPase and NFkB Western blots.\n8) DAPI (4',6-diamidino-2-phenylindole) is written with capital letters. Please define first and write this way.\n9) Please provide vendor information for DAPI.\n10) On Page 6: correctly it is called Delta-Delta Ct method.\n11) Part of the statistical analysis is missing, which needs to be added.\n12) Page 8, lines 1 and 110: The use of \u201cplacenta\u201d is not correct in this context.\n13) It would be useful if subheadings could be used in the Discussion.\n14) Reference is missing from the sentence ending on Page 10/line 13,\n15) Figure legends are uploaded twice.\n16) Figure 1: Please define \u201cNC\u201d.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines (v0.1)\". PeerJ https://doi.org/10.7287/peerj.153v0.1/reviews/2\nReview 5: Reviewer 3 \u00b7 Jun 7, 2013\nBasic reporting\nNo comments\nExperimental design\nThe study is very preliminary and completely descriptive. I have several concerns regarding the experimental design and the interpretations of the results.\nValidity of the findings\nNo comments\nAdditional comments\nIn this study the authors have investigated the effect of cannabidiol on two cell lines derived from human choriocarcinoma. The conclusions are based on descriptive data of mRNA and protein abundance. Nevertheless the results are discussed as if the changes observed would affect the placental barrier.\n\nMajor points:\n\n1. Previous studies have already shown the expression in BeWo and JAr cell lines of a complete panel of transporters and pumps, in addition to MDR1 and BCRP (see for instance Serrano et al., Placenta 28 (2007) 107-117). Several results described here as original (for instance page 7, lines 6-8) should be commented as mere confirmation of previous reports.\n\n2. Page 8, line 9. The present paper offer little clue on the mechanism of action of cannabidiol on the expression of these two ABC proteins. The fact that mRNA levels were consistent with protein abundance is an interesting data but adds little to the mechanism controlling the expression of these genes.\n\n3. Are the observed effects due to specific or non-specific (for instance chemical stress-mediated) mechanisms? Are cannabinoid receptors expressed in JAr and BeWo cells? Are they responsive to cannabidiol? What is the intracellular signalling pathway involved? Is there any evidence for a role of any nuclear receptor?\n\n4. Page 11, lines 15-22. This paragraph is highly speculative. Are the authors proposing an autocrine mechanism? What are the meadiators?\n\n5. Page 12, lines 8-13. The conclusion is again very speculative, above all, the last sentence.\n\n6. Figure 1. Why BCRP was not included in this part of the study?\n\n7. Figure 1. Immunolocalization of MDR1 does not seem to be at the plasma membrane of BeWo and JAr cells, but intracellular. Is BCRP also located intracellularly? How this fit the results from the western blot analyses?\n\n8. Does the treatment with cannabidiol change the subcellular localization of these proteins?\n\n9. Any functional study would be required to suggest that cannabidiol is able to change the role of these proteins in the placental barrier.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #3 of \"Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines (v0.1)\". PeerJ https://doi.org/10.7287/peerj.153v0.1/reviews/3\nReview 6: \n",
    "LLM_extracted_review": "['Review 1: The revision addressed all the criticism raised by the reviewers.\\n\\nReview 2: Some major technical issues (PCR, western) were raised by the reviewers. Functional significance of the observed changes need to be proven. Additional experiments are required.\\n\\nReview 3: \\n1. The structure of the \"Introduction\" should be rearranged to stress cannabidiol (CBD) as a promising candidate for clinical utilization.\\n2. Authors should explain if there is a chance to use CBD therapeutically in pregnancy.\\n3. The source of cells should be given more precisely, and studies where the cells were used should be referenced.\\n4. Citations regarding cell cultivation should be reconsidered as only one deals with JAr cell lines.\\n5. The name of the company producing the High capacity cDNA RT kit should be provided.\\n6. The word \"chronic\" when speaking about exposure should be omitted.\\n7. PCR analysis should be remade, using one housekeeping gene is not sufficient for accuracy.\\n8. Results describing expression of P-gp/BCRP in all cell lines used are inconsistent.\\n9. The description of results from protein level to mRNA is questionable; reconsider semi-quantitative analysis of protein expression.\\n10. The results in Figure 2 seem questionable; protein load in gel was not the same everywhere.\\n11. The western blots in Figure 3 cannot be used for semi-quantitative analysis.\\n12. Figure 4 is lacking a picture of the western blot.\\n13. Figure 5 must state how many biological replicates were used for the analysis.\\n14. Authors should discuss the opposing effect of CBD on P-gp and BCRP.\\n\\nReview 4: \\n1. Some periods are misplaced and need to be fixed.\\n2. The word \"polarizer\" should be changed to \"polarized.\"\\n3. A sentence on Page 2 needs rephrasing.\\n4. The word \"protein\" should be added after kDa on Page 2.\\n5. \"Trophoblast-like\" should be used instead of \"trophoblast\" cell lines.\\n6. \"h\" should be added after 24 and 48 on Page 4.\\n7. Antibodies used for the Na/K ATPase and NFkB Western blots should be described.\\n8. DAPI should be defined and written with capital letters.\\n9. Vendor information for DAPI should be provided.\\n10. The Delta-Delta Ct method should be correctly named.\\n11. Part of the statistical analysis is missing and needs to be added.\\n12. The use of \"placenta\" is incorrect in some contexts.\\n13. Subheadings could be useful in the Discussion.\\n14. A reference is missing from a sentence on Page 10.\\n15. Figure legends are uploaded twice.\\n16. \"NC\" should be defined in Figure 1.\\n\\nReview 5: \\n1. The study is very preliminary and completely descriptive.\\n2. Previous studies have shown the expression of a complete panel of transporters in BeWo and JAr cell lines; some results should be commented as confirmation of previous reports.\\n3. The paper offers little clue on the mechanism of action of cannabidiol on the expression of ABC proteins.\\n4. The observed effects should be clarified as specific or non-specific mechanisms.\\n5. A paragraph is highly speculative regarding an autocrine mechanism.\\n6. The conclusion is speculative, especially the last sentence.\\n7. BCRP was not included in Figure 1.\\n8. Immunolocalization of MDR1 does not seem to be at the plasma membrane; clarification is needed for BCRP.\\n9. The treatment with cannabidiol\\'s effect on the subcellular localization of these proteins should be discussed.\\n10. A functional study is required to suggest that cannabidiol can change the role of these proteins in the placental barrier.']"
}