Macrophage surface antigen‐1  has been implicated in the regulation of osteoclastogenesis. In the synovial tissues of patients with aseptic loosening after total hip replacement, CD11b was up‐regulated, which indicated that CD11b is closely involved in osteolysis around the prosthesis. We found that CD11b, but not CD18, promoted osteoclast (OC) maturation. Here, we show CD11b up‐regulated the levels of spleen tyrosine kinase (Syk), c‐Fos and nuclear factor of activated T cells, cytoplasmic‐1 (NFATc1), as well as the activity of extracellular‐regulated kinase (Erk), and as a result, osteoclast precursors (OCPs) differentiated and became tartrate‐resistant acid phosphatase (TRAP)‐positive. In addition, increased tumour necrosis factor‐α (TNF‐α) induced by ultra‐high molecular weight polyethylene (UHMWPE) particles up‐regulated the level of CD11b. Taken together, these findings suggest that CD11b is a positive regulator of osteoclastogenesis and that it functions by activating the Syk signalling pathway, while CD18 does not have the same effect. Total joint replacement surgery brings marked symptomatic relief with substantial functional improvement. However, prosthetic implants can also cause a variety of possible complications. While failure due to sepsis, fracture and dislocation has become relatively rare, particle‐induced osteolysis has been a major cause of aseptic loosening after total joint replacement. It is estimated that over 70% of implant failures are caused by aseptic loosening, and UHMWPE particles have been found to correlate with osteolytic lesions significantly ‐CD3‐NK1.1‐CD11b−/lowCD115+CD117+). The OCPs were added at a seeding density of 1 × 104 cells per cm2 to 6‐well, 48‐well and 96‐well culture plates, and bone slices  were added to a 96‐well culture plate in advance for bone resorption assays. The cultures were fed every 2 days with fresh α‐MEM medium containing M‐CSF  and soluble RANKL .OCPs were obtained from the spleens of 4‐week‐old C57BL/6J mice purchased from The Jackson Laboratory . Briefly, the spleens were cut into small pieces using surgical sterile scissors and compressed using the plunger of a 1‐ml syringe in circular motion against bottom of the petri dish, until no macroscopic pieces of the spleen were visible. The suspension of cells was filtrated through a 40‐μm nylon mesh  and transferred to a 50‐ml tube to which 8 ml of culture medium was added. The spleen cell suspension was centrifuged at 1000 r.p.m. for 5 min., after which the cell pellet was redissolved in 2 ml of red blood cell (RBC) lysis buffer . The suspension was then incubated for 10 min. on ice and washed two times by centrifugation at 1000 r.p.m. for 5 min. with 10 ml of medium. Flow cytometry was used for the identification of OCPs, and most of the cells were OCPs  were added to the medium at a concentration of 10 μg/ml after exchanging the medium Itgam shRNA  and empty lentivirus (Mock) were added to the medium  after OCPs were seeded in the plate.Lentivirus  carrying The medium was supplemented with TNF‐α (Sigma‐Aldrich) up to 100 ng/ml to stimulate OCPs for 15 min. before the addition of sRANKL TRAP‐positive cells were detected using a TRAP staining kit (Sigma‐Aldrich) according to the manufacturer's instructions. The number of TRAP‐positive cells (OCs) in 48‐well plate was determined at a magnification of 40× by a microscopy , and then, the mean number of TRAP‐positive osteoclasts per well of each group was calculated ® Premix Ex Taq™ II (TaKaRa). All data were analysed by the comparative Ct method  Total RNA was extracted from the cells according to the manufacturer's protocol for TRIZOL reagent . Briefly, for total RNA extraction, cells cultured in 6‐well plate were mixed with liquid nitrogen and treated with TRIZOL reagent, at a proportion of 1 ml TRIzol for each well, following manufacturer's instructions. 500 ng of total RNA was used for reverse transcription , and, then 80 ng of cDNA samples was amplified with SYBRCells or periostea were lysed in 1X RIPA . A BCA Protein Assay Reagent Kit  was used to measure protein concentrations. Total protein was transferred to nitrocellulose membranes (Bio‐Rad) and incubated overnight with primary antibody: rabbit polyclonal anti‐Syk, anti‐P‐Syk (phospho‐Y352), anti‐c‐Fos, anti‐Erk1/2, anti‐P‐Erk, anti‐NFATc1, anti‐CD11b and anti‐β‐actin . Blots were then incubated with horseradish peroxidase (HRP)‐conjugated secondary antibody  in blocking buffer. Quantification of the immunoblots was performed using Image J software  after exposed to X‐ray film, and results were normalized to β‐actin 2 chamber. Blood was collected from the heart, and serum was collected after centrifugation. Serum TNF‐α was detected with corresponding ELISA kit  according to the manufacturer's instructions.At 2 weeks after surgery, all of the mice were killed in a CO9 particles/ml UHMWPE particles were provided by Mr. Ernst Krendlinger, the manufacturer of Clariant . Ninety per cent of the particles were less than 9 μm in diameter, with diameters ranging from 0.08 to 11.13 μm (S.E.M. = 1.31 μm), with a mean size of 1.72 μm Itgam‐knockout  mice on a C57BL/6J background were purchased from The Jackson Laboratory. Twelve of the C57BL/6J mice received sham operation (sham control), and the intact periostea on the surface of calvaria were reserved. After implantation of 100 μl PBS on the calvarium, the incision was sutured. The rest of the twelve C57BL/6J mice (WT group) and Itgam‐KO mice (Itgam‐KO group) were treated with UHMWPE particles. Briefly, after anesthetization, a 1 × 1 cm2 area of calvarial bone was exposed overlying the calvaria, and the intact periosteum was carefully reserved. Mice from WT group and Itgam‐KO group received 100 μl of UHMWPE suspension. The incision was then sutured. Water and food were given ad libitum. 2 weeks after the operation, all animals were killed in a CO2 chamber Itgam‐KO group, periostea were cut‐off from the edge and carefully isolated from the calvaria using a tweezer; then, the periostea were used for Western blot. Mice calvaria from all of the three groups was used for histologic evaluation after a micro‐CT scan.Institutional approval was obtained for experiments about animals and specimens from human, and all experimental procedures involving animals and human specimens were in accordance with the institutional review board of The Fourth Military Medical University. Twenty‐four female 8‐week‐old C57BL/6J mice (wild‐type) and twelve female 8‐week‐old 2 chamber, periostea were harvested for Western blot assays and the blood was used to measure TNF‐α using a mouse TNF‐α commercially available enzyme‐linked immunosorbent assay (ELISA) kit.Similarly, twelve female 8‐week‐old C57BL/6J mice were randomly divided into two groups of six mice. The UHMWPE particles were implanted using the same procedures as described above: Briefly, in the sham control, the mice received 100 μl of PBS spread over the area, and the particles group received 100 μl UHMWPE particle suspension. After 2 weeks, all animals were killed in a COSynovial tissues from patients with aseptic loosening after total hip replacement and fracture around the hip were made into formalin‐fixed sections (4 μm thick). Immunohistochemical staining was used to detect CD11b, and sections with over 50% of the cells stained brown were determined to be CD11b‐positive expression.in vitro micro‐computed tomography  of the calvarial bone structure was performed on samples of mice in each group, and bone volume fraction (BVF) was used to measure the level of osteolysis High‐resolution An Olympus BX‐51 microscope  was used for osteolysis measurements. Sections were stained with haematoxylin and eosin (H&E) to determine the bone resorption area. In detail, at a magnification of 40x, bone resorption area of each section was measured by tracing the area of tissue including any bone resorption pits, and the area was recorded and calculated by the software . The mean resorption area of each section was calculated. TRAP staining was used for OC quantification. The image was oriented with the midline suture, which was 1.5 mm in diameter, in the centre of the field.spss 13.0  was used to perform statistical analysis. Significance was assessed by independent two‐tailed Student's t‐test or anova. All data in histograms of this study were presented as mean ± S.D. P‐values <0.05 were considered significant.Itgam mRNA levels were down‐regulated in lentivirus‐infected cells . . Fig. .Itgam‐KO group exhibited little expression of CD11b and showed a down‐regulation of the proteins involved in the Syk signalling pathway  of WT group was lower than Itgam‐KO and sham control (Fig. Post‐operatively, all of the mice recovered from anaesthesia with no wound healing problems or other complications observed, and the animals were killed after 2 weeks. Although UHMWPE particle implantation resulted in osteolysis in the skulls of WT group and ved Fig. A and C. ved Fig. E. One‐warol Fig. B. Additirol Fig. D and F.Aseptic loosening after total joint replacement is the major underlying cause of joint revision, and bone resorption caused by osteoclasts plays an important role in the development of this pathological process Chi‐square test result showed that specimens from patients with aseptic loosening exhibited up‐regulated CD11b, suggesting that CD11b significantly correlates with aseptic loosening. In the present study, in vitro results showed that anti‐CD11b antibody treatment had a negative impact on OC differentiation, while OCPs treated with anti‐CD18 antibody exhibited no significant reduction in OC formation rate. In our study of CD11b function in OCs using lentivirus, we found that Itgam silencing reduced the number of TRAP‐positive and multinucleate cells, which provides evidence for the role of CD11b in OC formation at the gene level. The loss of resorption function of OCs after treatment with anti‐CD11b antibody or lentivirus carrying Itgam shRNA likely indicates cells in a stage of undifferentiation. We also performed studies using UHMWPE particles, one of the most common and biologically relevant sources of wear debris in clinical practice in vivo experiments, bone resorption levels of WT group and Itgam‐KO group were significantly higher than sham control, indicating that the osteolysis models were successfully built. In addition, Itgam‐KO mice exhibited a marked inhibition of osteolysis and recruitment of osteoclasts under a burden of UHMWPE particles, which further reflected the profound involvement of CD11b in osteoclastogenesis.This study indicates that CD11b promotes osteoclastogenesis. Itgam led to down‐regulation of the levels of Syk, c‐Fos, NFATc1 and p‐Erk/Erk. Previous studies indicate that Syk is necessary for OC differentiation et al. Although the promotion of OC differentiation by CD11b was shown in our study, it is notable that we found no previous research showing the specific mechanisms underlying this effect. Our results demonstrate that CD11b acts as a positive regulator of OC differentiation through the downstream Syk signalling pathway. In the present study, neutralization of CD11b or knockout of In vitro assessment of blood neutrophils showed a significant up‐regulation of CD11b after TNF‐α administration via various signalling pathways. Hence, in consideration of this role for CD11b in osteolysis, TNF‐α is confirmed to exacerbate bone resorption through up‐regulation of CD11b.In addition, we observed that TNF‐α stimulation up‐regulated the expression of CD11b in OCPs, and the levels of P‐Syk/Syk, P‐Erk/Erk, c‐Fos, NFATc1 in TNF‐α‐treated OCPs were increased. Besides, mice with up‐regulated TNF‐α showed increased CD11b expression. A previous study showed that TNF‐α induced an inflammatory response through a CD11b‐dependent mechanism. Itgam shRNA decreased the OC formation rate, while CD18 neutralization had no significant effect on OC differentiation. Besides, neutralization or deficiency of CD11b caused decreased bone resorption activity of OCs. In addition, knocking out Itgam inhibited osteolysis and OC recruitment in mice under a burden of UHMWPE wear particles. We further suggest that it is through the Syk pathway that CD11b promotes OC maturation. The deficiency of CD11b down‐regulated the expression of Syk, c‐Fos and NFATc1 and Erk phosphorylation. In our study, TNF‐α was shown to be a positive regulator of CD11b in OCPs and promoted osteolysis. Treatment with TNF‐α up‐regulated CD11b in OCPs, and mice with up‐regulated TNF‐α showed increased CD11b expression in periosteal tissue. The present findings suggest a regulatory role for CD11b in OCs that may provide a therapeutic target in osteolytic bone diseases.In summary, our data suggest that CD11b promotes RANKL‐induced OC differentiation through the Syk signalling pathway. The high positive rate of CD11b expression in specimens from patients with aseptic loosening indicates the relevance of CD11b expression to particle‐induced osteolysis. Our experiments demonstrate that the neutralization of CD11b using a neutralizing antibody or the infection of a lentivirus carrying The authors report they have no conflicts of interest.


Nicotiana benthamiana with Bamboo mosaic virus (BaMV), a gene with downregulated expression was found involved in the infection cycle of BaMV. To uncover how this downregulated gene affects the accumulation of BaMV in plants, we used loss- and gain-of-function experiments. Knockdown of this gene decreased the accumulation of BaMV coat protein to approximately 60% in both plants and protoplasts of N. benthamiana but had no effect on Potato virus X and Cucumber mosaic virus infection. The full-length gene was cloned and revealed as an N. benthamiana nuclear-encoded chloroplast carbonic anhydrase (CA) and so designated NbCA. As compared with the accumulation of BaMV RNAs in NbCA-knockdown protoplasts, both plus- and minus-strand RNAs were reduced. We further fused NbCA with Orange fluorescent protein to confirm its localization in chloroplasts on confocal microscopy. However, transiently expressed NbCA in chloroplasts did not considerably increase BaMV accumulation. The addition of exogenous CA may not have any additive effect on BaMV accumulation because of the natural abundance of CA in chloroplasts. In an in vitro replication assay, the addition of Escherichia coli-expressed NbCA enhanced exogenous template level (re-initiation and elongation) but not endogenous template level (only elongation). These results suggest that NbCA is possibly involved in re-initiation step of BaMV RNA replication. Further analysis indicated that proton concentration could influence the endogenous and exogenous template activities. Hence, our results implied that NbCA could be playing a role in harnessing proton concentration and favoring the replicase with the re-initiation template.On inoculation of  Bamboo mosaic virus (BaMV), belonging to the Potexvirus genus of family Alphaflexiviridae  and bicarbonate (HCO3-). The reaction of CO2 + H2O ↔ HCO3- + H+ reaches equilibrium spontaneously but slowly and can be accelerated by the catalyzation of CA , antioxidant activities in response to pathogens  is a zinc metalloenzyme that can catalyze the interconversion of carbon dioxide  in disease resistance  and exhibits CA enzymatic, SA-binding, and antioxidant activities in sistance . In CA-sf the HR .N. benthamiana.Although this earlier research mostly documented that the host CA is necessary for plant defense, in this study, we found that CA could, by contrast, help BaMV accumulation. Therefore, we investigated how CA could play a role in assisting BaMV accumulation in plants. Furthermore, we studied whether CA is involved in the initiation step of BaMV replication in Nicotiana benthamiana plants were grown in a growth room at 28°C with 16 h light and 8 h dark. Three viruses were used for inoculation: BaMV strain S (Potato virus X (PVX) strain Taiwan, and Cucumber mosaic virus (CMV) strain NT9  was cloned into the pGEM-T Easy vector  in a previous study (ACAC10-1 in Tobacco rattle virus (TRV)-based VIGS  gene or phytoene desaturase (PDS) gene were used as a negative or positive control, respectively. Agrobacterium containing pTRV1, pTRV2-NbCA, pTRV2-Luc, or pTRV2-PDS was cultured at 30°C to OD600 0.8∼1.0; the cells were collected by centrifugation at 5000 rpm, then suspended in the induction medium  at 30°C for 1 h. After induction, equal volumes of both cultures  were mixed before agroinfiltration onto the second, third, and fourth leaf of true leaves of a 1-month old N. benthamiana. When the PDS-knockdown plants had a photo-bleach phenotype, 500 ng of BaMV virion was mechanically inoculated onto the fourth leaf above the infiltrated leaves.The cDNA fragment of us study . To use sed VIGS  in N. beNbCA was calculated by measuring the expression ratio normalized to the expression of actin between the NbCA-knockdown and control plants. Two sets of primers were used for the RT-PCR to amplify NbCA and actin gene expression: NbCA/F (5′-AGTGCATGTGGAGGTATCAAAGGT-3′)/NbCA/R (5′-GTCGACTACGGAAAGAGAAGG-3′) and actin/3′ (5′- GTGGTTTCATGAATGCCAGCA-3′)/actin/5′ (5′-GATGAAGATACTCACAGAAAGA-3′).The knockdown efficiency of N. benthamiana and viral RNA inoculation was described previously , boiled with the SDS sample buffer  for 5 min, separated on a 12% polyacrylamide gel containing 0.1% SDS, transferred onto a nitrocellulose membrane (PROTRAN BA 85 Schleicher and Schnell), and probed with primary antibody  and with the secondary antibody [affinity purified anti-rabbit IgG conjugated IRDye 800 (ROCKLAND)]. Finally, membranes with fluorescent bands were scanned by using LI-COR Odyssey (LI-COR Biosciences). In addition, rbcL (RuBisCo large subunit) stained with Coomassie blue was used as a loading control.4OAc, washed, dried, and dissolved in 30 μl de-ionized H2O. For RNA extracted from protoplasts, cells were mixed with 200 μl protoplast RNA extraction buffer . After phenol/chloroform extraction and ethanol precipitation, RNA was further precipitated with 3 M NH4OAc and dissolved in 13 μl de-ionized H2O.Total RNA was extracted from leaves with STE buffer , 1% SDS, and 3.3 mg/ml bentonite and an equal volume of phenol/chloroform. After ethanol precipitation, the RNA was further precipitated with 3 M NH2, 50 mM KCl, 0.1% Triton X-100, 2 μl cDNA, 3 units of Taq DNA polymerase (Promega) and RNase-free water to a final volume of 20 μl. Cycling conditions began with an initial hold at 95°C for 5 min, followed by about 30 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 30 s. Reactions were carried out in a RotorGene 3000  with data acquisition at 72°C on the channel, excitation at 470 nm and detection at 585 nm, by using a high-pass filter for both plus- and minus-strand. The reaction without template or reverse transcriptase was a negative control, and actin was detected for normalization. All samples were run at least three times.qRT-PCR was used to detect both BaMV plus- and minus-strand genomic RNA. The cDNA synthesis reaction involved use of ImProm-II Reverse Transcriptase  as instructed with the primers for Oligo dT(25T) and BaMV+51 (5′-ACTGCCAATTGTCCCCTACA-3′) for the plus- and minus-strand, respectively. For quantifying the accumulation of BaMV genomic RNA or minus-strand RNA, primers for BaMV+51 and BaMV-282 (5′-TGTGCTGAACGGGTTATGAG-3′) or BaMV+1766 (5′-CACATCCGGCACTTACCA-3′) and BaMV-2002 (5′-ATGTATCACGGAAATAAGAGTT-3′) were used, respectively, in the reaction containing a 1000X dilution of SYBR green I . qPCR was performed in 0.2-ml PCR tubes with 0.6 mM primer, 0.2 mM each deoxyribonucleoside triphosphate, 10 mM Tris-HCl pH 8.8, 1.5 mM MgClN. benthamiana was cloned into the pEpyon binary vector that carries the mOrange2 reporter gene . The PCR product was first cloned into the pGEM-T easy vector , then sub-cloned into pEpyon with BamHI and KpnI after sequence verification.The full-length CA cDNA of ter gene  to expreAgrobacterium containing the binary vector with NbCA-OFP or vector alone was cultured and infiltrated into N. benthamiana plants. The fluorescent signals were detected at 3 days post-infiltration by confocal laser scanning microscope . To observe whether NbCA altered its localization after BaMV infection, pKBG, a plasmid containing an infectious cDNA of BaMV with a GFP reporter . The PCR product was cloned into the pGEM-T easy vector and the sequence was verified. Finally, NbCA was subcloned from the T-vector into the pET29a(+) expression vector (Invitrogen) and transformed into E. coli BL21(DE3). The resulting clone was designated pET29a(+)-NbCA.The coding sequence of  peptide  was amplEscherichia coli containing pET29a(+)-NbCA was cultured to OD600 = 0.7 ∼ 1.2 , the expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16°C for 1 day, then samples were centrifuged at 7000 rpm at 4°C for 7 min. The cell pellet was resuspended with 8 ml buffer A  containing protease inhibitor cocktail  and subjected to the French Press to break cells: the sample was loaded into the French Press and squeezed out four times, then centrifuged at 12000 rpm at 4°C for 10 min. The supernatant was incubated with 1 ml complete His-tag Purification Resin  overnight, washed with 10 ml buffer A containing 50 mM imidazole, and eluted with buffer A containing 250 mM imidazole. Finally, the eluted protein was dialyzed with 150 ml buffer A four times to remove imidazole and stored at -80°C with the addition of final 10% glycerol. The vector-only construct was manipulated under the same condition as the negative control.Bamboo mosaic virus-infected leaves were collected at 5 dpi and homogenized with polytron in replicase complex extraction buffer  with a 2 ml/g of buffer/leaf ratio. The leaf slur was filtrated through Miracloth  and centrifuged at 500 × g for 10 min to remove the cell debris. The pellet was resuspended in suspension buffer  after centrifugation at 30,000 × g for 35 min. Approximately 2 ml of the extract was loaded on 28 ml of 20 ∼ 60% continuous gradient of sucrose with the gradient buffer  and centrifuged at 72,100 × g for 7.2 h. The 5th and 6th fractions of the 10 fractions (from top to bottom) with the highest RdRp activity were pooled and stirred with 1.5% NP-40 for 1 h to solubilize the membrane-associated RdRp.in vitro replication assay with the endogenous RNA templates, 15 μl of the replicase complex preparation (pH 8.0) was added to a total 50 μl reaction containing 2 mM  TP, 2 μM UTP, 3 mM MgCl2, 10 mM dithiothreitol, 50 mM Tris-HCl pH 8.2 , 12 unit RNase OUT , 8 mg bentonite, 0.066 μM [α-32P]UTP  and 5.2 μg recombinant NbCA at 30°C for 1 h . Therefore, the Tris-HCl buffer at different pH was used in the reaction to reach the final target pH as 7.1, 7.5, 7.8, 8.6, or 8.8, respectively. The reaction was stopped by adding 150 μl 5 mM EDTA, extracted with phenol/chloroform, and precipitated with ethanol. The radioactive RNA products were resolved on a 1% agarose gel and quantified by using the PhosphoImaging analyzer BAS-2500 (FUJIFILM).For the For the exogenous RNA templates, 15 μl of the replicase complex preparation was first treated with 10 units of micrococcal ribonuclease containing 2.5 mM Ca(OAc)2 to remove endogenous RNA at 30°C for 30 min in a total volume of 11.5 μl reaction. Then the reaction was terminated by adding 16 mM EGTA and set on ice for 1 ∼ 2 min. An aliquot of 15 μl mixture was subjected to a total 50 μl reaction as in the endogenous RNA template reaction. The radioactive RNA products were resolved on a 5% polyacrylamide gel and quantified by using the PhosphoImaging analyzer BAS-2500 (FUJIFILM).in vitro transcription with plasmids constructed previously , 40 mM Tris-HCl pH 8.0, 2 mM spermidine, 8 mM MgCl2, 10 mM dithiothreitol, 0.4 mM NTP and 200 U T7 RNA polymerase at 37°C for 2 h. The RNA was then gel purified, quantified, and stored at -80°C.Ba-77 RNA (the 3′-end 77 nt of BaMV minus-strand RNA) and r138/40A RNA (the 3′ UTR of BaMV RNA) was prepared in an eviously . The reaACAC10-1, found downregulated in N. benthamiana plants by cDNA-amplified fragment polymorphism (AFLP) after BaMV inoculation -based NbCA knockdown.The sequence of a gene, culation , showed NbCA expression in N. benthamiana did not cause any morphological change as compared with control plants (infiltrated with a TRV vector-carrying luciferase gene) . At 5 days post-inoculation (dpi), the accumulation of BaMV in NbCA-knockdown plants was reduced to 64% that in Luc-knockdown control plants . Hence, NbCA could be a positive regulator for BaMV infection in N. benthamiana.The reduced NbCA for BaMV accumulation is involved in virus replication or movement, cell wall-excluded protoplasts were prepared for viral RNA inoculation to eliminate the involvement of viral movement. The accumulation of BaMV coat protein in NbCA-knockdown protoplasts was reduced to 64 and 61% that of control protoplasts at 24 and 48 h post-inoculation (hpi), respectively . To determine whether the involvement of NbCA is specific to the BaMV infection cycle, CMV and PVX were inoculated into NbCA-knockdown protoplasts. The accumulation of the coat protein of these two viruses in knockdown protoplasts did not differ from that in control protoplasts at 24 and 48 hpi .To determine whether the role of NbCA-knockdown protoplasts was reduced by approximately 30 and 44% as compared with control protoplasts. The similar reduced ratio in both plus- and minus-strand of BaMV RNA was observed at 48 hpi . Therefore, the accumulation of plus- and minus-strand BaMV RNAs were similarly affected by the reduction in NbCA levels . These results suggest that NbCA is most likely involved in BaMV replication.Furthermore, to elucidate whether this deficiency results from a defect in synthesizing the plus- or minus-strand viral RNA, qRT-PCR was used to quantify the accumulation of BaMV RNAs in knockdown protoplasts. At 24 hpi, the accumulation of the plus- and minus-strand of BaMV RNA in NbCA full-length gene, a primer was designed for the 3′ rapid amplification of cDNA ends (RACE) experiment to obtain the downstream sequence of ACAC10-1. The cDNA fragment derived from 3′ RACE contains the stop codon of NbCA. The upstream sequence of ACAC10-1 including the start codon of NbCA was retrieved from the transcriptome of the N. benthamiana draft genome (NbCA coding region and cloned into the pEpyon binary vector (mOrange2 reporter gene (OFP), to produce the NbCA-OFP fusion protein. Furthermore, the amino acid sequence of NbCA (accession no.: MF346699) was aligned with those from N. tabacum , and Arabidopsis  . NbCA was mainly localized in chloroplasts. Moreover, to observe whether the localization of NbCA was altered after BaMV inoculation, we co-infiltrated the infectious BaMV viral vector pKBG carrying green fluorescent protein (GFP) driven by subgenomic RNA promoter .To visualize the localization of NbCA in plant cells, NbCA-OFP was transiently expressed in promoter  with NbCNbCA-knockdown plants  and protoplasts , NbCA may assist viral RNA replication. To validate this hypothesis, we transiently expressed NbCA-OFP in N. benthamiana followed by BaMV inoculation. However, accumulation of BaMV coat protein was not enhanced at 3 dpi. The pool of NbCA in cells may be enough for BaMV replication and the addition of exogenous NbCA by transient expression might not provide additional help for BaMV accumulation. Hence, we used in vitro replication (E. coli to acquire the purified-NbCA for in vitro replication experiments. The E. coli BL21 (DE3)-expressed recombinant NbCA-His was purified through a Nickel-chelating resin column .Since we found that the accumulation of BaMV coat protein and viral RNA was reduced in lication  to excluE. coli-expressed NbCA in the replication assay . Second, we tested whether NbCA is involved in the initiation of BaMV replication. In the in vitro replication, we tested the two RNA templates, r138/40A  .First, we tested whether NbCA affects endogenous RNA template activity, which represents the elongation step of BaMV replication. Viral RNA synthesis did not differ with or without the addition of the nthesis)  and Ba-7nthesis) . The addin vitro replication assay, CA affected the exogenous but not the endogenous template activities. If the CA activity provides the free proton to change the micro-environment (reducing pH) such as the membrane-housed viral replication site, the condition for the re-initiation of the plus- or minus-strand RNA temples by BaMV replicase complex could be regulated. To test this hypothesis, we used various pH conditions for in vitro replication assays with endogenous and exogenous templates. The endogenous template  favored a higher pH condition . By contrast, the exogenous template  favored a lower pH condition. These results are implying that CA might be trapped into the viral replication site to produce free protons to create a more acidic microenvironment favoring the re-initiation of viral RNA replication.Carbonic anhydrase activity condenses carbon dioxide with water to produce a free proton in the reaction. We wondered whether the proton concentration affects the viral RNA replication. In the 2 into bicarbonate for carbon fixation   . Even thigure 4) , CA posiNCE103, the Saccharomyces cerevisiae CA-like gene deletion strain sensitive to an oxidized environment such as in the presence of H2O2; hence, these two CAs were found to exhibit antioxidant activities  was demonstrated to play an critical role in the minus-strand RNA synthesis of BaMV and was also involved in anti-oxidation processes in cells . If NbCA is simply an antioxidant in general, it should favor both endogenous and exogenous templates in the in vitro replication assays. By contrast, the coat protein accumulation of CMV and another potexvirus, PVX, did not differ from that in the control . We assumed that chloroplast-localized NbCA would affect viruses that replicate in chloroplasts. CA is involved in various biological processes including SA binding . The replication complex associated with various host proteins including CA on the thylakoid membrane might create a replication competent microenvironment. Thus, the optimal condition for BaMV initiation and elongation could be regulated .Another possibility for NbCA assisting BaMV is fine-tuning the condition for viral RNA replication. Because BaMV replicates in chloroplasts and possibly associates with the thylakoid membrane in stroma . The pH + oxidoreductase (FNR). FNR transfers electrons from the reduced form of ferredoxin (Fd) to NADP+ and produces NADPH that consumes a proton with the reaction 2 Fdred + NADP+ + H+ → 2 Fdox + NADPH (One of the CA activities in general is converting one carbon dioxide into bicarbonate and releasing one proton, which might act on the replicase complex and change the proton concentration at the microenvironment level to initiate RNA synthesis. Once the initiation kicks in, a switch from initiation to elongation is needed to increase pH for efficient elongation by turning off the NbCA activity or using another factor to replace NbCA. A possible candidate that could reduce the proton concentration is ferredoxin-NADP + NADPH .We have identified a host factor that could assist in BaMV RNA replication. This factor, NbCA, could play a role in regulating the switch of initiation and elongation of RNA synthesis.I-HC and C-HT designed the research, analyzed the data and wrote the manuscript. AT, Y-PH, I-FW, and S-FC performed the experiments. Y-HH and C-HT participated in data analysis and discussion.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


White wine lees had high mDP content compared with red ones. Catechin (50–62%) and epicatechin contents were the predominant terminal units of polymeric proanthocyanidin extracted from examined samples. Epigallocatechin was the predominant extension unit of white wine lees, whereas epicatechin was the predominant compound in red wine marc. The ORAC  assay was strongly correlated with the DPPH  assay, and the results showed the strong antioxidant activities associated with red wine lees (PN > 35 mg Trolox/g FDM) . This study indicates that tannin is one of the major phenolic compounds available in wine lees that can be useful in human and animal health applications.The effect of vinification techniques on phenolic compounds and antioxidant activity of wine lees are poorly understood. The present study investigated the antioxidant activity of white and red wine lees generated at early fermentation and during aging. In this study, the total phenol content (TPC), total tannin content (TTC), mean degree of polymerization (mDP), and antioxidant activities of five white and eight red wine lees samples from different vinification backgrounds were determined. The results showed that vinification techniques had a significant ( Approximately 75 million tonnes of grape were produced annually, of which 85% is used in the wine industry, and around 9 million tons of organic waste is generated . These wWine lees consist of dead yeast, yeast residues or particles precipitated at the bottom of wine tanks or barrels . CurrentThe present study examined the characteristics of lees from a variety of sources, including different grape varieties and winemaking techniques, in order to improve understanding of this material and devise useful strategies to add value to this waste stream. Lees samples were characterized by determining total phenolic content, tannin content, mean degree of polymerization (mDP), and antioxidant activity of their extracts.Sodium carbonate, anhydrous di-sodium hydrogen orthophosphate, and ascorbic acid were from BDH . Methylcellulose, gallic acid, and phloroglucinol were purchased from Sigma Chemical Co. . Trolox , Folin–Ciocalteu reagent, α,α-diphenyl-β-picrylhydrazyl (DPPH), 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), catechin, and epigallocatechin gallate were obtained from Sigma-Aldrich Chemical Co. . Sodium dihydrogen phosphate monohydrate was purchased from LabServ Biolab . Ammonium sulfate was obtained from J.T. Baker . Ethanol (100%) was obtained from Fisher Scientific . Methanol (100%), acetic acid, and hydrochloride were obtained from Merck . All reagents and chemicals used in this study were of analytical grade or higher. The water used was produced from E-pure Barnstead water system Model no. 02622. The water used was obtained from E-pure Barnstead water system Model no. 02622 .Vitis vinifera). The white varieties were Sauvignon Blanc (SB), Chardonnay (C), and Pinot Gris (PG), and the red variety was Pinot Noir (PN). Winemaking encompassed a number of common techniques, such as natural fermentation, commercial yeast inoculation, maceration on skins, pump over, and hand plunging  and commercial wineries during March–April 2009. A total of fourteen wine lees samples were obtained from small-scale winemaking exercises which utilized six white and seven red wine lees samples  ethanol was added to 0.5 g of freeze-dried sample, and the sample was shaken using a thermostatic orbital shaker  for 60 min. Samples were then centrifuged at 3000× g for 5 min using a Megafuge 1.0 with a Siehe rotor . Supernatants were decanted, subsampled and stored at −20 °C. Wine lees and sample preparation were done in triplicate.Lees samples were extracted as described by Mercurio et al. . Five mLw/v) was added. The solution was mixed again and kept at room temperature for an hour. The absorbance of the mixture was read at 720 nm using a visible spectrophotometer  against a water blank. This analysis was performed with triplicate measurements of three subsamples for each extract. The concentration of total phenolic was calculated from a standard curve constructed using gallic acid solutions following the method described above. The range of gallic acid standard concentration was between 0 and 1 g/L. The total phenolic content was expressed as mg gallic acid equivalents (GAE) per gram of dry lees (mg GAE/g FDM).The total phenol content of wine lees extracts was determined as described by Makkar et al. . A 0.1 mThe total tannin content of extracts of freeze-dried wine lees was determined using the methylcellulose precipitable (MCP) tannin assay as described by Sarneckis et al. . This isThe mean degree of polymerization (mDP) of wine lees extracts was determined as described by Kennedy and Jones (2001) . The metFor acid catalysis, a 0.50 mL aliquot of blank reagent (0.2 M HCl in methanol containing 20 g/L ascorbic acid) or phloroglucinol solution (0.2 M HCl in methanol containing 100 g/L phloroglucinol and 20 g/L ascorbic acid) was added to 0.50 mL labelled control and phloroglucinol test tubes, respectively. All the samples and controls were incubated at 50 °C for 20 min, and the reaction was stopped by adding 5 mL of 40 mM aqueous sodium acetate solution prepared fresh .v/v aqueous acetic acid and 2% v/v acetic acid in methanol, respectively) were used and the eluting peaks were monitored at 280 nm. The flow rate was set at 0.8 mL/min, with the mobile phase set initially as 95% mobile phase A/5% mobile phase B for 5 min, followed by 90% mobile phase A/10% mobile phase B for 25 min, then 60% mobile phase A/40% mobile phase B for 30 s, and finally, 100% mobile phase B for 6 min, before equilibrating the column to the initial running conditions. To construct the standard curve, solutions A (1000 ppm catechin solution) and B (1000 ppm epicatechin solution) were made and used at concentrations of 0 to 100 ppm. The standard samples were processed as described above. To estimate the proanthocyanidin adducts, the response factors stated by [An Agilent HPLC 1100 series was used to analyze phloroglucinol adducts according to the method described by Kennedy and Jones (2001) [Antioxidant activity of wine lees extracts was determined using α,α-diphenyl-β-picrylhydrazyl radical (DPPH•) and oxygen radical antioxidant capacity (ORAC) methods.515) was measured at 0, 5, 10, 20, 30, and 60 min of reaction using a spectrophotometer against a water blank. The cuvettes were covered with parafilm and kept in a light-tight container between absorbance measurements.The ability of lees extracts to scavenge the DPPH• was evaluated using methods previously described by Sánchez-Moreno et al. , with moThe concentration of remaining DPPH• in the reaction medium at each reaction time was calculated from a calibration curve of DPPH• of absorbance at 515 nm (A515) against concentration. The equation below was applied to calculate the percentage of remaining DPPH• at the reaction time of 10 min. The percentage of remaining DPPH• was then plotted against weight (mg) of extract.50 . EC50 of different sample extracts were calculated from the regression equation.Results were also presented in ECThe ORAC assay is a widely used method to measure the antioxidant activity in medical and life sciences fields. It uses 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) as a peroxyl radical generator , and repFLUOstar Omega multifunctional microplate reader  was used to measure the fluorescein intensity. The instrument was set up to read fluorescence intensity mode. The excitation, emission, and cut off wavelength were 485 nm, 538 nm, and 530 nm, respectively, and the gain was adjusted to 85%. The measurement was taken over 60 min at 1 min intervals.Diluted antioxidant standard and samples (25 μL) were added to the 96-well microplate, followed by the addition of 150 μL of 10 nM fresh fluorescein working solution to each well. The microplate was then covered by parafilm and incubated at 37 °C for 30 min. After incubation, 25 μL of the AAPH (65 mg/mL) solution was automatically injected into each well by the plate reader pump followed by 5 s shaking.The area under the curve (AUC) method was used for quantification of ORAC . AUC of blank and antioxidant was calculated from the equation below:p-value < 0.05 was tested by application of Tukey’s multiple comparisons test. Error bars in graphs indicate standard deviation (SD) of the mean. Pearson’s correlation coefficients among the measured parameters were also calculated. The analysis was Genstat® Software  was used for statistical analysis.Analysis of variance (ANOVA) was carried out to investigate the effects of samples (dependent variable) on the measured parameters. Significant difference at p < 0.05) than lees from other white varieties . This may be explained by the processing of PG wine where a maceration step is used during processing (The average TPC of the wine lees extracted with 50% ethanol is shown in ocessing . In addip > 0.05) from both PNR1 and PNR2.The TPC of lees from rosé wine (PNR1 and PNR2) was not affected by pre-fermentation maceration period . This is consistent with the results of Sacchi et al. , which dSaccharomyces cerevisiae [The TPC of red wine lees varied widely (P < 0.05) depending on the processing conditions and geographical location of the samples. PN lees from Central Otago with no cap management (PN6) had the highest TPC (41.0 ± 1.6 mg/g FDM), which was more than twice the level found in Waipara Pinot Noir macerated for 1-week pre-fermentation . A “cap” is formed when crushed grapes are pushed to the surface of must by yeast-generated carbon dioxide during fermentation. “No cap management” means a cap was allowed to form, and the cap formation was not disturbed, which would allow trapping the heat generated during the fermentation process and an increase in the ferment temperature would result. The rise of fermentation temperature (above 15 °C) is advantageous to the growth of revisiae , and canThe effects of maceration on PN is observed in red wine lees samples in which PN3 with 1-week pre-fermentation maceration had a lower TPC than PN1 (no maceration). Pinelo et al.  reportedp < 0.05) compared to hand plunged (PN5) technique. This observation is consistent with Sacchi et al. (2005) that varying TPC was obtained in the wine that is treated with “pumped over” compared with “hand plunged” techniques. Moreover, the addition of oak wood chips (PN4) may lead to a high TPC in wine lees. Therefore, the TPC of wine and any by-products (such as lees in the present case) will vary depending on the vinification techniques used, and the presence of certain chemicals, such as sulfur dioxide [The technique used for increasing the release of phenolics in wine may have an impact on the TPC in lees. For example, Central Otago PN lees from pumped over wine (PN2) had higher TPC . However, more tannins were obviously present in red lees compared with white and rosé wines lees (p < 0.05) maybe because the tannin extractability in PN3 was limited by applying 1 week pre-fermentation maceration with an insufficient amount of SO2. TTC of PN6 was significantly greater than PN2 and PN5 (p < 0.05). This may indicate that rise of temperature during fermentation allowed the extraction of more tannins than pump over (PN2) and hand plunged (PN5) techniques. The high TTC of PN4 might be due to the contribution of oak chips added in maceration. According to Gao et al. [Total tannin content (TTC) of the wine lees samples is shown in nes lees . The simnes lees . The TTCo et al. , the exto et al. . TherefoThe mean degree of polymerization (mDP) of proanthocyanidins extracted from seven wine lees sample was determined by phloroglucinol analysis. The mDP values of red wine lees were low, ranging from 8.7 ± 3.2 to 12.7 ± 5.0 . By contThe monomer composition (%) of terminal and extension units in the breakdown of polymeric proanthocyanidin were calculated . The resBordiga et al.  and Lee 50 is a measurement of capacity to scavenge DPPH radicals which are expressed as the amount of dry lees required to scavenge 1 g DPPH radicals. Extracts of red wine lees exhibited the highest activity (EC50 ranged from 12.6 to 17.7 g FDM/g DPPH), followed by PNRs, and PG (EC50 ranged from 33.6 to 35.9 g FDM/g DPPH), and white wine lees extracts exhibited the least activity (EC50 ranged from 40.6 to 78.2 g FDM/g DPPH). The DPPH• data are also presented as a percentage of DPPH remaining over 60 min of reaction to demonstrate the efficacy of scavenging the radical . About 15 mg of Chardonnay lees was required to reduce DPPH radicals in the assay by 50% within ten minutes. The same level of inhibition was achieved after 20 min with 7.50 mg of lees. Only 7.50 mg of SB samples, 3.75 mg lees of PG and PNRs, were required to achieve 50% reduction within ten minutes. PN wine lees showed a much stronger free radical scavenging activity, with only 0.938 mg of samples required to achieve 50% reduction. While the scavenging of DPPH radicals was concentration-dependent, PN lees showed a different behaviour where the percentage of DPPH remaining at 7.50 mg of lees was higher than the ones with 1.875 and 3.750 mg of lees. The higher amount of lees addition leads to an early termination of the reaction. Villaño et al. [The antioxidant activities of the wine lees extracts were evaluated through their capacity to scavenge DPPH and AAPH radicals . EC50 iso et al.  reported50 = 0.026 g/g DPPH) compared with many other phenolic compounds. Therefore, lees macerated with oak chips will have higher gallic acid content and have a better antioxidant activity similar to wine aging in oak barrel or macerated with oak chips.While sample PN4 had lower TPC and TTC than PN6, a similar level of antioxidant activity was found in these two samples. As mentioned earlier, maceration with oak chips for samples PN4 can lead to higher gallic acid content which can change the composition of the phenolics in the extracts without a significant increase in the TPC. Larrauri et al.  and Vill50) of lees extracts (PN) in current study ranged from 4361.1 ± 20.3 to 6225.1 ± 96.2 mg extract/g DPPH. In comparison with the finding in PN and Pinot Meunier (PM) wine wastes reported by Cheng (2011), the antioxidant activity (EC50) of PN lees extracts are similar or higher than skin (EC50 ranged from 616.4 to 9606), and lower than pomace and seed .The results of ORAC assay was expressed as the capacity of equivalent Trolox (mg TE/g FDM) to scavenge AAPH radicals. The results are in agreement with the results of DPPH radicals scavenging activity, where PN1, PN4, and PN6 showed the highest antioxidant activity. Results from the present study show that PN wine lees contained high phenolic compounds and antioxidant activities, and therefore, it is a useful source of antioxidants. The vinification techniques can affect the TPC and TTC, and consequently, their biological activities. In addition, the results may indicate that tannin is one of the major phenolic compounds that contribute free radical scavenging abilities.The mDP values of white wine lees were higher than red ones, and this might be attributed to the source of proanthocyanidin, oxidation, and anthocyanin in red wine. Catechin was one of the predominant terminal units of polymeric proanthocyanidin found in both white and red wine lees. Epicatechin was also a major terminal unit of polymers in lees. Epigallocatechin and epicatechin were the predominant extension units of white and red wine lees, respectively. The above results indicate that red wine lees is a very rich in phenolics with high antioxidant activities, and can potentially be an easier material to extract phenolics from, unlike grape seed and skin, which require several drying and size reduction processes before extraction. Lees has a great potential to be used as a source of bioactive compounds.


Controlling contractile instabilities in the actomyosin cortex. Published 24, January 2017In Materials and Methods, we found a conflict and typo in strain information.What was referred to in the materials and methods section as strain number SWG010 should be corrected to SWG003. This strain (SWG003) was first published in Naganathan et al Elife 2014.nmy-2(cp8[nmy-2::GFP + unc-119(+)]) I; unc-119(ed3) III; gesIs002[Ppie-1::Lifeact::tagRFP-T::pie-1UTR + unc-119(+)], for imaging of GFP labelled NMY-2 .SWG003: What was referred to in the materials and methods section as strain number SWG011 should be corrected to SWG012.nmy-2(ges6[nmy-2::tagRFP-T + unc-119(+)]) I; xsSi5[cb-UNC-119 (+) GFP:: ANI-1 (AH+PH)] II; unc-119(ed3) III, for imaging of tagRFP-T labelled NMY-2 and GFP labelled AHPH for a probe of active RhoA in the cortex .SWG012: The article has been corrected accordingly.


The influenza virus RNA genome exists as a ribonucleoprotein (RNP) complex by interacting with NP, one of virus-encoded RNA binding proteins. It is proposed that trimeric NP is a functional form, but it is not clear how trimeric NP is formed and transferred to RNA. UAP56, a cellular splicing factor, functions as a molecular chaperone for NP and is required for the replication-coupled RNP formation of newly synthesized viral genome, but the details of NP transfer to viral RNA by UAP56 is unclear. Here we found that UAP56 is complexed with trimeric NP, but not monomeric NP. Gel filtration analysis and atomic force microscopy analysis indicated that the complex consists of two trimeric NP connected by UAP56. We also found that UAP56 stimulates trimeric NP formation from monomeric NP even at physiological salt concentrations. Thus, UAP56 facilitates the transfer of NP to viral RNA since trimeric NP has higher RNA binding activity than monomeric NP. Further, UAP56 represses the binding of excess amount of NP to RNA possibly by transferring trimeric NP. Collectively, we propose that UAP56 stimulates viral RNP formation through promotion of the assembly of trimeric NP and is important for the structural integrity of NP-RNA complex. NP is one of the basic viral proteins and binds single-stranded RNA without sequence specificity. NP is essential to maintain the RNA template in an ordered conformation suitable for viral RNA syntheses6. Cryo-electron microscopy analysis revealed that the oligomerization of NP is important to form a double helical structure with anti-parallel strand of vRNP8. Oligomeric NP shows higher RNA binding activity than monomeric NP9. Although crystal structures of RNA-free NP showed that NP forms oligomers in the crystalline state, a broad size distribution was observed by gel filtration chromatography in solution at physiological salt concentrations12, suggesting that NP exists in an equilibrium between monomers and oligomers including trimers and tetramers13. Thus, an exact form of NP to be assembled into vRNP remains unknown.The genome of influenza type A viruses is single-stranded RNAs of negative polarity. The viral genome (vRNA) exists as ribonucleoprotein (designated vRNP) complexes with heterotrimeric viral RNA-dependent RNA polymerases and nucleoprotein (NP)15. NP chaperones are also required to suppress the aggregation of NP. UAP56 is a cellular splicing factor belonging to the DExD-box family of ATP-dependent RNA helicase16. It is reported that the newly synthesized viral genome is co-replicationally assembled into RNP complex by UAP563. UAP56 binds to NP free of RNA but not NP-RNA complexes15. It is also reported that UAP56 interacts with N-terminal region of NP, and NP interacts with C-terminal region of UAP5615. However, the molecular mechanism of the transfer of NP to RNA by UAP56 is still unknown.For efficient viral transcription and replication, not only viral factors but also host factors are required. It has been reported that RAF-2p48/NPI-5/UAP56, Tat-SF1, and Prp18 function as molecular chaperones for NP to recruit NP to the viral RNAsHere we examined the binding stoichiometry of NP-UAP56 complex using recombinant UAP56 and RNA-free NP. We found that UAP56 stably interacts with trimeric NP, and stimulates the assembly of trimers from monomeric NP. Gel filtration experiments showed that the molecular size of UAP56-NP complex is more than 440 kDa, suggesting that the complex is composed of 6 molecules of NP and 2 molecules of UAP56. By atomic force microscopy (AFM) analysis of NP-UAP56, we found a dumbbell-shaped complex which is considered as two trimeric NP connected by UAP56. We also revealed that UAP56 facilitates NP-RNA complex formation without excessively oligomerized NP-RNA complexes. Taken together, these results suggest that UAP56 stimulates the RNP formation through the assembly of trimeric NP and controls the amount of NP on RNA for the structural integrity of NP-RNA complex for efficient viral RNA synthesis.E. coli BL21(DE3) strain and purified by Ni-NTA resin and GST resin, respectively . Plasmids expressing His-tagged NP and UAP56 were constructed using pET14b and pGEX-2T as previously described15. For the construction of a plasmid expressing R416A NP mutant, cDNA containing R416A point mutation was amplified from pET14b-NP by overlap PCR with primers 5ʹ-TCAGTACAGGCAAATCTCCCT-3ʹ, 5ʹ-AGGGAGATTTGCCTGTACTGA-3ʹ, 5ʹ-GGGAATTCCATATGGCGTCTCAAGGCACCA-3ʹ, and 5ʹ-CCGGTCGACTAGTGATGATGATGATGATGGCCATTGTCGTACTCCTCTGCATTGTCTCCG-3ʹ. The cDNA was cloned into pET14b at Nco I and Nde I sites.Rabbit anti-UAP56 antibody was prepared as previously describedPlasmids expressing NP-His, R416A NP-His, and GST-UAP56, were transformed into BL21(DE3) strain (Stratagene), respectively. The proteins were expressed in LB medium for 16 h at 18 °C after induction with 0.2 mM IPTG. For NP-His and R416A NP-His, the harvested cells were suspended in lysis buffer A  containing 0.1% Triton X-100 and 1 mM PMSF. After lysis by sonication, the lysates were centrifuged at 20,000 × g for 30 min. The supernatants were filtrated with a 0.45-μm filter and subjected to His-tag purification using Ni-NTA agarose beads . After washing with five column volumes of buffer A, NP proteins were eluted in buffer A containing 300 mM imidazole. To remove the bacterial RNA bound to NP, the NP lysates were treated with RNase A before purification and washed with buffer A containing 1.5 M NaCl during purification. Purified NP with a ratio of the absorbance at 260 nm to that at 280 nm of between 0.57 and 0.59 was used for this study. For GST-UAP56, the cell pellet was resuspended in lysis buffer B  containing 0.1% Triton X-100 and 1 mM PMSF. After sonication, the lysates were centrifuged at 20,000 × g for 30 min. The supernatants were filtrated with a 0.45-μm filter and subjected to GST-tag purification using glutathione Sepharose beads . After washing with 5 column volumes of buffer B, GST-UAP56 immobilized on the resin was digested with an appropriate amount of PreScission protease for 12 h at 4 °C. UAP56 without GST tag was collected in the flow through fraction.After buffer exchange of purified NP and UAP56 to buffer C  using a desalting column , purified NP and UAP56 were mixed at a 1:1 molar ratio, and were incubated at 4 °C for overnight. The NP-UAP56 complex was purified by a gel filtration chromatography using the HiPrep 16/60 Sephacryl S-200 HR column  on AKTA system  or Superose 6 PC 3.2/30 column on SMART system (Amersham Pharmacia) in buffer C. The SDS-PAGE gel of NP-UAP56 was visualized by either CBB staining or VisPRO™ 5 Minutes Protein Stain Kit . The band intensities were measured by ImageJ software.19.NP alone and NP-UAP56 complex were subjected to AFM analysis in air at room temperature . The system was operated in tapping mode with a 100-μm scanner. Probes made of a single silicon crystal with a cantilever length of 129 µm and spring constant of 33–62 N/m  were used for imaging. Data were collected in the height mode. Images were captured in 512 × 512 pixels and the captured images were flattened and plane-fitted before analysis. The size of the particles was calculated with a correction for tip effect as previously reported32P-labeled 53 nt-long model viral genome (5′-AGUAGAAACAAGGGUGUUUUUUCAUAUCAUUUAAACUUCACCCUGCUUUUGCU-3′) or 165 nt-long model viral genome (5′- AGUAGAAACAAGGUCGUUUUUAAACUAUUCGACACUAAUUGAUGGCCAUCCGAAUUUAUGGUCCACGGUGGUUUUUGUGAGUAUCUCGCGGGUGCGAGACUGCGACAUUAGAUUUCUUAGUUCUUUUAUUCUUUCCAUAUUGAAUAUAAUUGACCUGCUUUCGCU-3′) in a buffer containing 50 mM HEPES-NaOH (pH 8.0), 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 5% glycerol at 30 °C for 30 min. The samples were applied to a 6% native PAGE in 0.5 × TBE buffer and visualized by autoradiography.Increasing amounts  of NP or NP-UAP56 complex were incubated with 0.5 pmol of2, 1 mM DTT, and 5% glycerol. The samples were separated on 6% native PAGE in 0.5 × TBE buffer, and the gels were subjected to western blotting using anti-UAP56 and anti-His-tag antibodies, respectively.UAP56  or NP-UAP56 complex  was incubated with 0.5 pmol of 53 nt-long model vRNA in a buffer containing 50 mM HEPES-NaOH (pH 8.0), 100 mM NaCl, 5 mM MgClSupplementary Figure


Millepora platyphylla is an important component of Indo-Pacific reefs, where it thrives in a wide range of environments, the ecological and biological processes driving its distribution and population structure are not well understood. Here, we quantified this species’ population structure in five habitats with contrasting hydrodynamic regimes in Moorea, French Polynesia; two in the fore reef: mid and upper slopes, and three in the lagoon: back, fringing and patch reefs. A total of 3651 colonies of fire corals were mapped and measured over 45,000 m2 of surveyed reef. Due to the species’ sensitivity to fragmentation in response to strong water movement, hydrodynamic conditions  corresponded to marked differences in colony size distributions, morphology and recruitment dynamics among habitats. The size structure varied among reef habitats with higher proportions of larger colonies in calm nearshore reefs (fringing and patch reefs), while populations were dominated by smaller colonies in the exposed fore reefs. The highest densities of fire corals were recorded in fore reef habitats (0.12–0.20 n.m-2) where the proportion of recruits and juveniles was higher at mid slope populations (49.3%) than on the upper slope near where waves break (29.0%). In the latter habitat, most colonies grew as vertical sheets on encrusting bases making them more vulnerable to colony fragmentation, whereas fire corals were encrusting or massive in all other habitats. The lowest densities of M. platyphylla occurred in lagoonal habitats (0.02–0.04 n.m-2) characterized by a combination of low water movement and other physical and biological stressors. This study reports the first evidence of population structure of fire corals in two common reef environments and illustrates the importance of water flow in driving population dynamic processes of these reef-building species.While the fire coral  Millepora hydrocorals, also called fire corals, are an important component of reefs communities worldwide where they, similar to scleractinian corals, contribute to reef accretion and community dynamics [Millepora species also contribute to the survival of corals during Acanthaster outbreaks as this corallivorous predator tends to avoid Millepora species [Coral reefs exhibit a remarkable diversity of organisms that reside within highly variable environments resulting in strong spatial variability in species’ distribution patterns . For scldynamics ,7. Fire dynamics  and colodynamics . Fire codynamics  and oftedynamics ,12. On t species .Hydrodynamic forces in the form of water-displacement, velocity and acceleration have been recognized as a key factor in determining the shape and occurrence of many reef-building organisms –16. In cWater flow can drive the spatial distribution in adult populations through the distribution and dilution of larval settlement cues  and dispPocillopora damicornis and Acropora hyacinthus, often grow into large and delicate arborescent colonies in areas of relatively high water flow [Water flow also influences colony growth and morphology ,33. Undeter flow ,39,40, bter flow . Asexualter flow ,43. Fireter flow ,9,36,44.Millepora platyphylla, Hemprich & Ehrenberg 1834, the only species of fire coral found in French Polynesia [M. platyphylla were conducted in five habitats on the north shore of Moorea  with differing amounts of water flow: fore reef habitats with high water movement, especially on the upper slope and decreasing with depth to the mid slope. Lagoonal habitats  are sheltered from waves and oceanic swell, except during storms, and water movement in these habitats is less than on the fore reef [M. platyphylla among reef habitats can be attributed to different flow regimes.In this study, we investigated whether and how different reef habitats with contrasting water regimes affect the population structure of olynesia ,46. Survore reef ,17. We eMillepora platyphylla is a gonochoric broadcast spawner that reproduces sexually by producing medusoids and planula larvae [M. platyphylla can also reproduce asexually through fragmentation [a larvae . The meda larvae . M. platentation ,9.M. platyphylla growing within fore reef habitats are exposed to lower wave energy on the mid slope compared to the those growing on the upper slope, near where the waves break. In the lagoon, the wave energy disperses from the reef crest towards nearshore reefs [Between April and December 2013, a series of surveys were conducted on the north shore of Moorea, French Polynesia, in the South Pacific Ocean , at four different locations . Five habitats with contrasting water flow regimes were selected; two in a fore reef environment: the mid slope (13 m depth) and the upper slope (6 m depth), and three in the lagoon (< 1 m depth): the back reef, fringing reef and patch reef . These h2 reef area being surveyed. The north shore of Moorea is ~16 km long with a fore reef area of ~3.15 km2 and a back reef with a hard-bottom area of ~4.58 km2 [2 on the fore reef, which is ~0.1% of the total fore reef area on the north shore of Moorea. We also performed six belt transects of 0.003 km2 on the back reef area (i.e. both back and patch reef habitats), which is ~0.07% of the total back reef area. All colonies of M. platyphylla that were at least 50% within the transect borders were measured, photographed and georeferenced using SCUBA. No specific permit was required at the time of field work for sampling protocols described herein and our surveys did not involve endangered/protected species and did not require animal tissue/skeleton collection.Within each habitat, three 300 m long by 10 m wide belt transects were laid over the reef parallel to shore, at least 30 m apart, resulting in a total of 45,000 m4.58 km2 . We perfM. platyphylla colonies were georeferenced by determining their position along the transect-line (0 to 300 m) and straight-line distance from both sides of the transect (0 to 10 m). From these measures, each colony was mapped with x and y coordinates, from which the distribution index (DI) and mean neighborhood distance (ND) were calculated using the spdep package [-2) and cover (%) were also calculated for each transect (i.e. 3000 m2). Using these variables, variability in the spatial distribution among habitats was quantified by one-way PERMANOVA tests in PRIMER 6 software [M. platyphylla, we assumed that swell wave energy exposure decreases with habitat depth and its proximity to the coastline, as demonstrated in previous studies [r coefficient was used to test for significant correlations.All  package  in R [52 package  and calc package . The measoftware , since a studies ,47. ConsM. platyphylla populations were generated from estimates of colony sizes computed from 2D photographs. Photographs were taken from above the colony and included a plate of known dimensions positioned next to each colony. For bigger colonies, pictures were taken from a larger distance, and for 5 colonies (out of the 3561) 2 photographs were required to photograph the whole colony. Each colony size, standardized as the projected surface, was then measured (in cm2) using ImageJ 1.4f software [1), indicative of the relative abundance of small and large colonies within a population. CV and g1 were computed for each habitat per transect together with standard descriptive statistics, such as 95% percentile of the mean  and the2 were considered as recruits and were most likely the result of sexual reproduction. Larger colonies with a size between 1 and 20 cm2 were classified as juveniles based on previous studies on coral recruitment using both settlement plate experiments [2 based on previous studies on other reef-building taxa [The mean abundance and proportion of recruits, juveniles and adults were estimated for each transect whereby the three life stages were defined based on colony size. Colonies with a total size (surface) below 1 cmeriments  and fieleriments . While ting taxa . Differe2 were removed from this analysis to only retain the mean height and size of adults for each transect. Adult colonies were assigned to one of these three morphologies: 1) encrusting: thin colonies growing against the substratum , was recorded and linked to the colony size data previously described. Colonies below 20 cmbstratum ; 2) “shebstratum  and 3) mbstratum . DiffereM. platyphylla among the five surveyed habitats, i.e., those on the fore reef (mid and upper slopes) and those in lagoonal habitats .Similarities in population structure based on the following parameters: density, cover, DI, ND, mean adult colony size and height, and proportion of recruits, juveniles and adults were calculated and visualized using a hierarchical complete-linkage agglomerative clustering (CLUSTER) method and a non-parametric multidimensional scaling (MDS) ordination on normalized data in PRIMER 6 software. Multivariate PERMANOVA on aforementioned characteristics was used to determine differences in population structure of M. platyphylla was found in all habitats, but its population composition differed among habitats. A total of 3651 colonies of M. platyphylla were counted in the five surveyed habitats. Most colonies (48.2%) occurred on the upper slope, whereas M. platyphylla colonies on patch reefs accounted only for 5.2% of all colonies (P < 0.01) and was higher on the upper slope  and mid slope , i.e. fore reef habitats, compared to lagoonal habitats   and was again highest on the upper slope . Colonies occurred closer together on the mid slope (6.64 ± 1.86 m) and upper slope (4.09 ± 0.34 m) where the average distance among neighboring fire coral colonies was 4.3 times smaller compared to lagoonal habitats  (P < 0.01).colonies . Colony N = 189) . M. plat2 and approximately one third (30%) of aforementioned colonies fell in recruit and juvenile size classes, i.e. were smaller than 20 cm2. The size-frequency distributions of M. platyphylla populations differed among certain habitats, but were similar among the lagoonal fringing and patch reefs with populations dominated by a combination of small  and large colonies (≥ 2050 cm2) resulting in bimodal size-frequency distributions (P < 0.05). All fire coral populations were characterized by relatively symmetrical size distributions (g1: –0.01–0.71), but the degree of skewness was again lower on the fringing and patch reefs (2) and back reef (2512 cm2) compared to other populations   . With 64 cm2) 2, , colonie cm2) 2,  of all hP < 0.05 and P < 0.01, respectively) with 96% of all recruits and juveniles occurring in fore reef habitats (48% for both the mid and upper slopes) and only small numbers were observed in lagoonal habitats (P < 0.01). Recruits were found in low proportions in most habitats with the highest value recorded on the mid slope (3.4 ± 0.4%), while no recruit was observed on the back reef  and 1094 juveniles (30%) were observed within the five surveyed habitats . The abuhabitats . The fraack reef ; Fig 5. ack reef ; Fig 5. ack reef , that waack reef . No signM. platyphylla colonies ranged in size from 0.18 cm2 to 189 062 cm2 (projected surface) and 0.1 cm up to 130 cm in height. Mean colony size and height of adults  differed among habitats . Fire corals were approximately 4 times larger on average in the upper reef slope (2308 ± 115 cm2), fringing (1590 ± 329 cm2) and patch reef (3090 ± 1294 cm2), compared to colonies growing in the back reef and mid slope . Massive morphologies dominated nearshore reefs (fringing reef: 79.7 ± 8.3% and patch reef: 59.0 ± 9.9%) whereas colonies were mostly encrusting on the mid slope (79.9 ± 1.1%) and back reef (74.5 ± 5.2%) ( ± 5.2%) . On the M. platyphylla varied significantly among reef habitats . Based on MDS, two main clusters can be distinguished: one with populations from wave exposed fore reef habitats, i.e. mid and upper slopes, and a second cluster consisting of populations from lagoonal habitats, i.e. back, fringing and patch reefs . Fractions of recruits and juveniles increased with increasing depth , while total cover decreased . Colonies grew in an encrusting morphology at mid depth and in the sheet tree morphology in shallow waters , where wave energy was higher and colonies mostly occurred in the encrusting morphology .Using all variables of each transect surveyed within the fore reef habitat (i.e. six replicates), we found that adult colonies became smaller (i.e. colony size decreases) with increasing depth   , we sougM. platyphylla. Water flow is also considered as an important factor influencing a colony’s morphology [Millepora that was only observed in low proportions in all other habitats (0–9%).Although differences in size-frequency distributions among habitats were found, e.g. few larger colonies in the calm fringing and patch reefs, smaller colonies in the mid slope and medium size colonies in the back reef, the degree of skweness was similar among all habitats with populations consisting of both small and large colonies. These distributions likely reflect low mortality in small size classes, as well as the persistence of the larger ones . Our resrphology ,71, generphology ,35. ThisM. platyphylla recruits and juveniles could not be related to adult population size in fore reef habitats. The proportions of recruits and juveniles were highest at mid-depths (13 m) on the fore reef where wave energy and solar irradiance are lower compared to the shallower depth habitats (as described in [M. platyphylla (3.2%) occurred on the upper slope where wave breaking first occurs, i.e. wave energy is the highest. Many studies investigating spatial distributions in coral reef communities often find that high energy reef zones restrict species’ distributions and cover [M. platyphylla shows the opposite trend: we observed high density and cover in the upper slope, a high energy reef zone, where colonies are growing in a contagious pattern of distribution. Such differences in fire coral distribution patterns in habitats of high energy are mostly related to the wave-vulnerable sheet tree morphology of Millepora. This growth form occurred nearly exclusive on the upper slope, while colonies were massive or encrusting in other habitats. The unusual sheet tree morphology observed in the upper slope has been described as a successful strategy exploited by Millepora to preempt the space and to compete with other coral taxa [M. platyphylla can rapidly overtake newly available space through clonal propagation and stolonal spreading may explain the increase of fire coral cover on Moorea’s reefs following the massive decline in coral cover from the Acanthaster outbreaks and cyclone Oli in 2010 [M. platyphylla’s cover was stable at approximately 1% at 6 m on the fore reef, i.e. more than 3 times lower than in 2013 at the same location. On the other hand, fragmentation usually induces corals to regress in size and increases mortality, especially in small size classes [Millepora allows them to reach larger colony sizes. This study shows that asexual reproduction through fragmentation and stolonal spreading likely plays a key role in structuring M. platyphylla populations where water flow is high and where fire corals face wave-induced breakage.Fire corals, like many other reef-building organisms, reproduce through both asexual and sexual reproduction with a dimorphic life cycle, with a pelagic dispersive phase (i.e. medusoids and larvae), followed by a sessile adult phase ,8. If diribed in ). Low waribed in . At shalribed in , but alsribed in . The aburibed in . Such phribed in . Howevernd cover ,78. M. pral taxa ,36. Waveral taxa , while tral taxa . The fac in 2010 . Between classes . Here, tMillepora species, is strongly determined by the local density of adults [M. platyphylla. In the lagoon, fire corals are characterized by wave-tolerant morphologies (i.e. encrusting and massive) suggesting that asexual reproduction through colony fragmentation is less likely of structuring importance compared to fore reef habitats. Colonies on the fringing reef, where wave energy is typically low, were distributed in patches. In Moorea, the fringing reef is exposed to large waves in the austral summer [M. platyphylla observed.In the lagoonal environment, the wave energy is reduced by the reef crest  likely ef adults ,82, and f adults  and/or sf adults ,76. The f adults –86, larvf adults , larval f adults  and settf adults . Poorer f adults ,91. The f adults . These df adults  and coull summer , which h2 (recruits and juveniles), a higher fraction than observed for 14 different genera of scleractinians corals in Moorea . The maintenance and recovery of fire coral populations are foremost sustained by the growth of remnant colonies, local recruitment through sexual reproduction where wave energy is low and clonal propagation in high wave energy zones.It must be noted that abundance of recruits and juveniles was likely underestimated in this study as they are difficult to find due to their small size during field surveys. Still, we identified 32% of colonies below 20 cm29%, see ). The ab29%, see  and the 29%, see . Our resS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


APOBEC3G is a cytidine deaminase with two homologous domains and restricts retroelements and HIV-1. APOBEC3G deaminates single-stranded DNAs via its C-terminal domain, whereas the N-terminal domain is considered non-catalytic. Although APOBEC3G is known to bind RNAs, APOBEC3G-mediated RNA editing has not been observed. We recently discovered RNA editing by the single-domain enzyme APOBEC3A in innate immune cells. To determine if APOBEC3G is capable of RNA editing, we transiently expressed APOBEC3G in the HEK293T cell line and performed transcriptome-wide RNA sequencing. We show that APOBEC3G causes site-specific C-to-U editing of mRNAs from over 600 genes. The edited cytidines are often flanked by inverted repeats, but are largely distinct from those deaminated by APOBEC3A. We verified protein-recoding RNA editing of selected genes including several that are known to be involved in HIV-1 infectivity. APOBEC3G co-purifies with highly edited mRNA substrates. We find that conserved catalytic residues in both cytidine deaminase domains are required for RNA editing. Our findings demonstrate the novel RNA editing function of APOBEC3G and suggest a role for the N-terminal domain in RNA editing. A3G has homologous NTD and CTD but only the CTD is active for deamination of ssDNAs242425711142627ssDNA was believed to be the substrate for the A3 family of enzymes6NMT1, CHMP4B, MAPK1, ACIN1, MED1, NFAT5, RBM14 which are previously known to be involved in HIV-1 replication, assembly, transcription and infectivity. Moreover, our findings suggest that the conserved residues of the zinc dependent deaminase motif of A3G-NTD are required for A3G-mediated RNA editing.To determine if A3G is capable of RNA editing, we transiently overexpressed the protein in 293T cells, a model routinely used by various labs to study A3G function and its mode of HIV-1 restriction5323334To determine whether A3G edits RNAs, we transfected 1 μg of pA3G into 293T cells , confirmed its expression  and perfAnalyses of RNA-Seq data identified single-nucleotide sequence variations in RNA sequences between the two groups of A3G and control transfectants for 712 genomic positions . At all HCFC1 and IGF2BP1. Two and 48 genes respectively had 3 and 2 editing sites . Editing levels were >20% and >30% for respectively 86 (12%) and 15 (2%) sites . 690 97. C>U eding sites . RNA eding sites . RNA eding sites  which caAmong the three empty vector control and three A3G overexpressing samples of the study, 18,028 genes were considered as expressed and were analyzed for differential expression. Of the 7,582 (42.1%) genes that were differentially expressed (P < 0.05), 61 and 83 were respectively down- and up-regulated with ≥2 fold-change in the A3G transfectants compared to the controls . CorrelaC[AG] , 179 (25%) and 163 (23%) of the 712 editing sites. We previously noticed that Cs edited by A3A are frequently flanked by inverted repeats3030To identify common features of sequence contexts of the editing sites, we examined 12 nt sequences flanking the edited C residue. There is a C for 613 (87%), U for 84 (12%), A for 9 or G for 6 sites at the immediate 5′ position of the edited C. This observation and sequence logo analysis  suggestsC[AG]  as a seqSCD, TM7SF3, CLASP1, PRPSAP2) to be informative in site-directed mutagenesis studies (below) or they were previously linked to HIV-1 infectivity . We validated RNA editing for 21 of 21 sites (100%) by Sanger sequencing in duplicate 293T/A3G transfectants  or cause missense changes in evolutionarily conserved amino acids  predicting altered protein function by at least one of three analysis programs  that was expressed in 293T cells and purified via affinity chromatography on anti-DDK column  216 nt MED1 RNA substrate that has 2 nt sequence variation (85 nt downstream from the edited C) from the endogenous MED1 RNA and can be selectively PCR-amplified without co-amplification of endogenous RNA. We incubated purified A3G in the presence or absence (control) of the ART MED1 RNA. Surprisingly, we detected A3G substrates KIAA1715, MED1, ITFG1, SCD, RFX7, CHMP4B, GOLGA5 and CLASP1 as well as A3A substrates SDHB, VIM, ASCC2 and TMEM179b in our control samples that contained recombinant A3G only  and vif-binding non-catalytic residues (D128K and P129A)263335SCD . PRPSAP2 reduced . Mutatio reduced . Our resIn this study, we show that transient expression of A3G in 293T cells causes site-specific C>U RNA editing of hundreds of genes . Sanger Previous studies of A3G ruled out any catalytic deamination activity of the A3G-NTD2639To determine the requirement for both A3G-NTD and CTD catalytic domains in RNA editing, a structure of the A3G holoenzyme, preferably bound to the ssRNA substrate is imperative. The crystal structure of the full-length A3G or the WT A3G-NTD is not available. However, crystal structures and solution NMR structures of A3G-CTD bound to the ssDNA substrate4243254749C sites, as previously observed with ssDNA templates, and the edited Cs are flanked by 3–10 base long pair of inverted repeats in 98% of the sites , APOBEC1 and A3A are all single-domain deaminases®, San Diego, CA). Each library, indexed for multiplex sequencing of six libraries per flow lane, was prepared from 1 μg of DNAse I-treated total RNA  after ribosomal RNA depletion with Ribo-Zero™ Gold reagents. Ten PCR cycles were used during library generation and the modal library fragment size was 300 bp. Paired-end, 101 nt sequencing of libraries was done on HiSeq™ 2000 instrument with TruSeq™ SBS and PE Cluster v3 Kit reagents (Illumina®). CASAVA 1.8.2 (Illumina®) was used for base-calling and de-multiplexing to obtain raw sequencing reads. Raw read data was deposited in NCBI BioProject with accession number 261741. Reads were filtered and trimmed to remove adapter sequences and poor-quality bases using Trimmomatic 0.33 with options: HEADCROP:12 ILLUMINACLIP: TruSeq3-PE-2.fa:2:30:10:6:TRUE LEADING:5 TRAILING:5 SLIDINGWINDOW:4:15 MINLEN:30. Raw and processed read counts are provided in Directional RNA sequencing libraries were prepared using the TruSeq™ Stranded Total RNA Sample Prep Kit (IlluminaReads were uniquely mapped to the GRCh38 human reference genome assembly (Ensembl release 78 of December 2014) using the Subread1.4.5-p1 aligner, with these options specified for the subjunc command: -d 30 -D 400 -uH. Reads were also uniquely mapped with the Tophat 2.0.12 aligner, with these options specified for the tophat2 command: –library-type = fr-firststrand –mate-std-dev = 35 -g 1 -N 3 –no-novel-juncs -r 10 –read-edit-dist 3. Genome indices for these aligners were respectively built with Subread index (options: -BF) and Bowtie2 2.2.3 bowtie2-build commands. Transcriptome index for Tophat was built with tophat2 command using Ensembl’s gene model for the genome assembly. RSeQC 2.4 was used to identify UCSC gene features of genomic regions that the reads mapped to. Mapping statistics are provided in The clipOverlap command (option: –poolSize 10000000) of bamUtil master version of 21 September 2014 was used to clip sequence overlaps between read pair-mates in mapped read data. The mpileup command (options: -AB -d 2000 -q 1 -Q 20) of Samtools 0.1.19 was then used to obtain genome-wide read base information from the mapped sequencing data. Custom scripts were used to parse the mpileup output into base calls. Genome positions at which the total A/C/T/G base call was <6 in any of the six samples or <9 in any of the two sample-groups of this study were ignored. Positions were then identified as possibly variant if all samples of at least one sample-group had ≥1 non-reference base calls of the same base-type at a variation level ≥0.02, with group-wide averages of ≥1.3 and ≥0.03 respectively. Variation level was calculated as the ratio of variant base call count to the sum of variant and reference base call counts. Variant positions for which calls for base-types other than the reference and variant constituted ≥1% of all A/C/T/G calls or were >1 in any sample were ignored. Positions with range/variation value of <2 for variation levels across all six samples were also ignored. A two-tailed beta binomial test (IBB 13.06 package for R 3.0.2) comparing variation levels of the two sample-groups was then performed for each of the remaining variant positions. P values obtained with the test were corrected for multiple testing with the Benjamini-Hochberg method. Positions for which the average variation level was >0 for both sample-groups or was <0.04 for ≥1 sample-group, or for which the corrected P value was ≥0.05, or which were not seen with both Subread- and Tophat-aligned data or failed a strand-bias test were ignored. The strand-bias test used base call counts across the group of A3G transfectant samples, and was applied to only those positions for which there were ≥8 calls for each of reference or variant base-types, with <25% of base calls of either base-type from reads of either forward or reverse direction. Failure was deemed if P value in a Fisher exact test that compared reference and variant base calls from reads of the two directions was <0.05. Finally, putative A3G-affected RNA editing sites were identified from the remaining genome positions for which the average editing level for the three A3G trasfectant samples was >0.05. The editing level was the mean of variation levels calculated from Subread- and Tophat-aligned data. The directional nature of the RNA sequencing libraries was used to identify the transcribed chromosomal strand at a genomic position to assign the type of RNA editing. RNA sequences flanking an editing site were deduced from the reference human genome and corrected for homozygous single-nucleotide polymorphisms in the HEK-293T cell-line. The 22 March 2015 release of ANNOVAR tool with its RefSeq-based hg38_refGene database was used to annotate sites with information on gene, gene feature and effect of editing of protein sequence. The list of editing sites with annotations is provided in PCR amplification and Sequencing primers (Integrated DNA Technologies) are listed in Sequence-verified plasmid constructs in pCMV6 vector for CMV promoter-driven expression of human A3G cDNA, with sequences matching NCBI RefSeq sequences NM_021822.1, for the generation of C-terminal Myc-DDK-tagged A3G proteins were obtained from OriGene . Site-directed mutagenesis of A3G constructs . Thawed 293T cells were passaged at least twice before transfecting the cells at ~50–60% confluency with plasmid DNA using jetPRIME . Transfection efficiency was 60–80% as assessed by fluorescent microscopy of cells transfected with the pLemiR plasmid DNA (Open Biosystems) for expression of a red fluorescent protein. Cells were harvested 2 days after transfection.M-PER reagent  with 1X Halt protease and phosphatase inhibitor cocktail (Thermo Fisher) was used to prepare whole cell lysates. Reducing and denaturing polyacrylamide gel electrophoresis of 20 μg proteins in Laemmli buffer system was performed on pre-cast, 4–15% gradient polyacrylamide gels . Following electrophoresis, proteins were transferred to polyvinylidene difluoride membrane with a pore-size of 0.2 mm for 7 min at 1.3 A in a Bio-Rad Trans-Blot Turbo apparatus. Membranes were incubated in Tris-buffered 0.15 M NaCl of pH 7.5 with 0.05% v/v TWEEN 20 (TBS-T)  and 5% w/v dried, non-fat, cow milk  for an hour at room temperature. After blocking, the membranes were incubated with primary antibodies overnight at 4 °C. Since A3G is MycDDK tagged at the C-terminus, we used mouse monoclonal anti-DDKKIAA1715 ORF RNA of 405 nt sequence and MED1 ORF ART RNA of 216 nt were generated by in vitro transcription using cDNA obtained from 293T cells as template and were amplified using KIAA1715-RNA(+T7)-F/KIAA1715-RNA-R primers and MED1-T7-WT-F/MED1-M1-R1 primers, respectively (in vitro transcription as per the manufacturer’s instructions to generate KIAA1715 and ART MED1 RNAs. RNA that was isolated from the transcription reaction was treated with DNAse I (Thermo Fisher). The RNAs were purified using RNA clean-up and concentration kit  and its integrity was verified by electrophoresis on an agarose gel. Recombinant human A3G (rA3G) or ADAR containing a C-terminal Myc-DDK tag  was purchased from OriGene . A3G protein expressed in 239T cells was purified on anti-DDK affinity column. In vitro assays with rA3G contained 0.84 μM A3G (approximately 2 μg), 50 pg KIAA1715 RNA/ART MED1 RNA, 10 mM Tris (pH 8.0), 50 mM KCl and 10 μM ZnCl2 (Sigma Aldrich). The reactions were incubated for 2 h at 37 °C. RNA was purified from the reactions using TRIzol (Life Technologies) as per the manufacturer’s instructions. Experiments containing rA3G or ADAR only contained 0.5 μM of the proteins (KIAA1715 or MED1-M1-R2 primer as the reverse primer for specific amplification of the ART MED1 RNA and subsequently sequenced using the T7 promoter primer (ectively . The forproteins . RT-PCR r primer .E. coli uracil DNA glycosylase (New England Biolabs) in 10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM DTT and 1 mM EDTA in a volume of 10 μl.1 μl of 1 N NaOH was added to the reaction, which was then incubated at 37 °C for 15 min. After adding 1 μl of 1N HCl and 12 μl of 2X loading buffer , the samples were incubated at 65 °C for 5 min. 10 μl of the reaction was electrophoresed on a 10% Mini-PROTEAN TBE-Urea gels (Bio-Rad). Typhoon 9400 Imager  was used to scan the gel in fluorescence mode at 488 nm.2 μM of 5′ Alexa Fluor 488 fluorescent dye-labelled ssDNA substrate of 40 bases (5′-ATTATTATTATTATTATTATTCCCAGGATTTATTTATTTA-3′) (Integrated DNA Technologies) was incubated at 37 °C for an hour with 100 nM or 1 μM r A3G (Origene) that was either pre-treated or untreated with Ribonuclease A (Sigma Aldrich) and 2 units of One-way ANOVA test followed by multiple comparisons is used to examine effects of various A3G mutations on RNA editing levels. GraphPad Prism 7.00 was used to perform statistical analysis and draw graphs.http://www.pantherdb.org). The edgeR 3.10.2 Bioconductor package was used for differential gene expression analyses. Only those genes with read counts >20 in at least three of the study’s six samples were analyzed; the rowsum.filter and prior.df values for estimateCommonDisp and estimateTagwiseDisp functions were respectively set at 12 and 0.2; and, P values calculated by the Fisher exact test were corrected with the Benjamini-Hocheberg method. Differentially expressed genes with a ≥2-fold change are noted in Sequence logos were created with the WebLogo 3 online tool. Gene set enrichment analyses were performed with PANTHER 9.0 online tool (URL: http://genetics.bwh.harvard.edu/pph2/), Mutation Taster (http://www.mutationtaster.org/) and Mutation Assessor (http://mutationassessor.org/r3/) softwares. Evolutionary conservation of edited amino acid residues is examined using UCSC genome browser (https://genome.ucsc.edu/).Functional effects of amino acid recoding by RNA editing are predicted by PolyPhen-2 .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.