SMAD1 and SMAD5, but reduced levels of the inhibitory SMAD7. Functional assays of GC B cells revealed that BMP-7 suppressed the viability-promoting effect of CD40L and IL-21, but had no effect on CD40L- and IL-21-induced differentiation into plasmablasts. BMP-7-induced apoptosis was counteracted by a selective TGF-β type I receptor (ALK4/5/7) inhibitor, but not by a selective BMP receptor type I inhibitor. Furthermore, overexpression of truncated ALK5 in a B-cell line counteracted BMP-7-induced apoptosis, whereas overexpression of truncated ALK4 had no effect. BMP-7 mRNA and protein was readily detected in tonsillar B cells, indicating a physiological relevance of the study. Altogether, we identified BMP-7 as a negative regulator of GC B-cell survival. The effect was counteracted by truncated ALK5, suggesting greater complexity in regulating BMP-7 signaling than previously believed.Selection and maturation of B cells into plasma cells producing high-affinity antibodies occur in germinal centers (GC). GCs form transiently in secondary lymphoid organs upon antigen challenge, and the GC reaction is a highly regulated process. TGF-β is a potent negative regulator, but the influence of other family members including bone morphogenetic proteins (BMPs) is less known. Studies of human peripheral blood B lymphocytes showed that BMP-6 suppressed plasmablast differentiation, whereas BMP-7 induced apoptosis. Here, we show that human naïve and GC B cells had a strikingly different receptor expression pattern. GC B cells expressed high levels of BMP type I receptor but low levels of type II receptors, whereas naïve B cells had the opposite pattern. Furthermore, GC B cells had elevated levels of downstream signaling components  FH) cells, exerts a powerful role in promoting differentiation to plasma cells and to induce CSR [FH cells provide support for GC B cells, also in the form of ligands for important co-receptors such as CD40 [Germinal centers (GC) are specialized structures that form transiently in secondary lymphoid organs following exposure to T cell-dependent antigens, and are the sites of B-cell maturation and selection. In the GC, the B cells’ immunoglobulin (Ig) genes undergo somatic hypermutation (SHM) and class switch recombination (CSR) to improve the affinity for antigens and gain specific effector function , 2. SHM duce CSR –9. Toget as CD40 , 11. In  as CD40 –14.ID1, ID2 and ID3 [The TGF-β family is a large superfamily of cytokines involved in regulation of proliferation, differentiation and apoptosis in different tissues and developmental stages. One subgroup of the TGF-β superfamily is the bone morphogenetic protein (BMP) subfamily containing 21 different cytokines . BMP sig and ID3 .Various BMPs can affect B-cell development at different stages . BMP-4 hTonsils were obtained from Agroklinikken , with written informed consent in accordance with the Declaration of Helsinki and the Regional Committees for Medical and Health Research Ethics, Region Eastern Norway (approved protocol REK#2010/1147a). The tonsils were processed to single cell suspension by mincing and stored as aliquots in liquid nitrogen. Peripheral blood was collected from anonymous, healthy donors at The Blood Bank in Oslo, after informed consent and with approval from regional authorities (REK S-03280). The human cell lines Jurkat, Sudhl-6 and Mino were from DSMZ . The HK cell line was a kind gift from Dr. Choi, Ochsner Clinic Foundation, New Orleans, USA. All cell lines were sustained in RPMI 1640 (PAA Laboratories) supplemented with 10% fetal calf serum (FCS) and streptomycin / penicillin (PAA)), but Mino cells were cultured in X-VIVO 15 serum free media  during functional studies.The following primary antibodies were used: biotinylated anti-ActRIIa (BAF340), -ActRIIb (BAF339), -BMPRII (BAF811),—ALK2 (BAF637), -ALK3 (BAF820), -ALK4 (BAF222) -ALK5 (BAF 3025), -ALK6 (BAF505), biotinylated goat IgG (BAF108) . The following antibodies were from BD Biosciences : Anti-active caspase-3 Alexa647 (Clone: C92-605), anti-CD20 APC-H7 (Clone: L27), -IgD PerCP-Cy5.5 (Clone: IA6-2), -CD38 PerCP-Cy5.5 (Clone: HIT2), -CD38 PE-Cy7 (Clone: HB7), -CD38 FITC (Clone: HIT2), -CD3 V500 (Clone: UCHT1), -CD27 APC (Clone: M-T2701), -CD44 APC (Clone: G44-26), and biotinylated anti-CD44 (Clone: G44-26). Anti-IgD FITC , -CD3 PE (Clone: UCHT1) and CD3 FITC (Clone: UCHT1) , anti-p-Smad 1/5 PE (Clone: D5B10), -p-Smad 2/3 PE (Clone: D27F4) and -p-ERK Alexa 647 (Clone: 197G2), -Smad 1 (#9743S), -Smad 4 (#9515) , biotinylated anti-CD38 (Clone: HIT2) , anti-β-actin (clone: I-19) (Santa Cruz Biotechnology), and anti-BMP-7 . The following secondary antibodies were used: rabbit anti-GAPDH . Biotinylated antibodies were detected using Streptavidin PE or Streptavidin APC . CD40L and Enhancer for Ligands (ALX-850-064) were acquired from Alexis Biochemicals, Enzo Life Sciences . IL-21 (PHC0214) was purchased from Invitrogen  and from eBioscience. BMP-2, BMP-4, BMP-6 and Activin A were purchased from R&D Systems . BMP-7 was purchased from R&D Systems and from BioLegend . The ALK2/ALK3 inhibitor LDN193189 (#S2618) and the ALK4/ALK5/ALK7 inhibitor SB431542 (#S1067) were purchased from Selleckchem .SMAD1 FAM (Hs00195432_m1), SMAD5 FAM (Hs00195437_m1), SMAD4 FAM (Hs00929647_m1), SMAD6 FAM (Hs00178579_m1), SMAD7 FAM (Hs00998193_m1) and PGK-1 VIC (4326318E), 1D1 (Hs03676575_s1), ID2 (Hs04187239_m1), ID3 (Hs00954037_g1), GAPDH FAM (4352934E), BMP7 FAM , PGK-1 FAM (4333765F).The following probes were from Applied Biosciences : 5 cells/ml) were cultured in 48-well plates containing irradiated HK cells seeded 24 hours in advance . Primary B cells were cultured in RPMI with 10% FCS, together with rhCD40 ligand (CD40L)  at 0.25 μg/ml , according to manufacturer’s recommendations.Tonsillar GC B cells, naïve and memory B cells , BMP-4 (25 ng/ml), BMP-6 (250 ng/mL) or BMP-7 (500 ng/mL) or as specified. RPMI with 10% FCS was used as culture medium, unless otherwise specified.- GC B cells were obtained through negative selection by combining Biotin Binder Dynabeads  with biotinylated anti-CD44 (BD Pharmingen) (1:50). A naïve/memory joint population was isolated using Biotin Binder Dynabeads (Thermo Fisher) with biotinylated anti-CD38 (eBioscience) (2.35 μg/mL). Cells and Dynabeads were incubated in the dark at 4°C with rotation. IgD-depleted memory B cells were obtained by negative selection by incubating CD19+ B cells with Pan Mouse IgG Dynabeads (Thermo Fisher) coated with mouse anti-human IgD Abs (BD) for 30 min at 4°C, followed by removal of beads.Tonsillar B cells were isolated using negative isolation with CD3 Dynabeads . CD3-depleted cells were split in two populations and incubated with antibodies for 10 min. CD44+IgD-CD27-CD3-CD38+), naïve (CD20+IgD+CD27-CD3-CD38-) and class switched memory B cells: (CD20+IgD-CD27+CD3-CD38-). Intracellular phospho-flow cytometry was performed on fixed cells , Electron Microscopy Sciences, PA, USA), permeabilized with 90% methanol and stained at room temperature for 60 min. All flow cytometry data were analyzed using the online Cytobank software (www.cytobank.org). Differences in protein expression are shown as arcsinh transformation, calculated as arcsinh (median fluorescence intensity (MFI) of activated cells/cofactor) subtracted by arcsinh (MFI of unstimulated cells/cofactor), using cofactors set to ≥ 150.Cells were labeled with antibodies and incubated at 4°C for 30 min. FACS analysis and sorting was carried out on a FACSCantoII and FACS Aria Flow Cytometer (BD), respectively. Using the anti-IgD FITC, -CD20 APC-H7, -CD27 APC, -CD3 PE and -CD38 PerCP-Cy5.5. B-cell subsets were obtained as specified: GC (CD20TGFBR1 (ALK5) and ACVRIB (ALK4), we designed our constructs as previously done with SMAD7 [TGFBRI, with an in frame KpnI site in order to fuse them with 2A_GFP in a pENTR vector (Invitrogen) as previously described [ACVRIB, we created a 2A_mCherry construct in pENTR by amplifying mCherry and PCR-fusing it to 2A with a KpnI site in the same frame as the GFP construct. This was done using the following primers: KI2AmCherr_F CAC CGG TAC CAG AGC CAA GAG AGG CAG CGG CGC CAC CAA CTT CAG CCT GCT GAA GCA GGC CGG CGA CGT GGA AGA GAA CCC TGG ACC AAT GGT GAG CAA GGG CGA G and mCherrSTOPERI_r TTG AAT TCT TAC TTG TAC AGC TCG TCC ATG. The full length sequence of human TGFBR1 (ALK5) and the truncated version  were designed based on the NCBI Database sequence (accession number: NM_004612). These constructs were codon optimized and synthesized by Eurofins . The ACVRIB coding sequence was PCR extracted from pDONR223-ACVR1B which was a gift from William Hahn & David Root (Addgene plasmid # 23567). We subcloned the amplicon (truncated ACVRIB) using the InFusion kit  with the following primers: (Alk4f) CCG CCC CCT TCA CCG ATG GCG GAG TCG GCC GGA G and tAlk4r (truncated) CCT CTC TTG GCT CTG gcC TTG TCT TTG GAG AGA C where the nucleotides in lower case are added to keep the frame of the fused vector (pENTR-mCherry). After sequence verification (Eurofins), the pENTR constructs were recombined to a Gateway compatible retroviral expression vector, pMP71-Gateway [To overexpress th SMAD7  where thescribed . For ACV-Gateway .Viral particles were produced as described previously . BrieflyViability was assessed by incubating cells with propidium iodide . Apoptosis was measured on fixed cells (1.6% PFA) and permeabilized with methanol (≥ 90%), followed by staining with anti-active caspase-3 antibodies . Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was determined by the In Situ Cell Death Detection Kit  as described by the manufacturer. Samples were analyzed on FACS CantoII (BD).SMAD expression was measured in duplex (except SMAD1) with PGK-1 VIC as endogenous control. Assays , or from Sudhl-6 as specified.RNA was isolated using RNeasy Plus kit  and cDNA was synthesized using the Quantitect kit (Qiagen), adding 10 ng of total RNA per sample. Quantitative real-time PCR (qPCR) was carried out on a 7500 Real Time PCR System (Applied Biosciences). Cells were lysed and processed for SDS-PAGE. Mini Protean or Criterion TGX precast gels were used for SDS-PAGE , transferred to PVDF membranes and hybridized with the indicated antibodies, followed by HRP-conjugated secondary antibodies. Protein bands were visualized using Amersham ECL Plus  or Pierce ECL-2 (Thermo Fisher) with Hyperfilm (GE) or SuperSignal West Pico and Dura (Thermo Fisher Scientific) or Clarity (Bio-Rad) with Chemidoc MP (Bio-Rad) applied for imaging. Image processing was performed by use of ImageLab (Bio-Rad) and Quantity One software (BioRad).Naïve and GC B cells from tonsils were fixed in 4% PFA  in PBS, washed in PBS and permeabilized in 90% methanol at -20°C. After washing in PBS, TUNEL staining (Sigma-Aldrich) was performed as described by the manufacturer, before cytospin samples were prepared using Shandon Cytospin 2 mounted in fluorescent mounting medium (Dako) containing Hoechst 33258 and visualized in a Zeiss LSM710 confocal unit , equipped with a 25x/0.8 oil objective. All images were taken randomly from the Hoechst staining. Images were exported as tiff images and assembled in Illustrator .t-test or a two-tailed unequal variance Student’s t-test as specified. Statistical differences were corrected for multiple testing.Statistical significance was determined by applying either a two-tailed paired Student’s BMP6 and BMP7 mRNA in GC B cells [-CD20+CD38+IgD-), naïve B cells (CD3-CD20+CD38-IgD+CD27-), plasmablasts (CD3-CD38hi) and three distinct memory B-cell subsets  (We have previously demonstrated expression of  B cells . In ordeB cells) . This apB cells) . In contB cells) . PlasmabB cells) . These rSMAD5 than naïve B cells, and also expressed SMAD1 which was undetectable in naïve and memory B cells . Naive, memory and GC B cells had detectable levels of ID1, ID2 and ID3 genes, and ID3 was significantly higher in naive B cells than GC B cells  and higher cell number yields than FACS sorting . Westernin vitro cultured GC B cells. As these cells are highly prone to apoptosis, a co-culture system featuring the FDC-like HK cell line was introduced to improve viability [BMP4, endogenous BMP expression levels were low or undetectable , while BMP-7 in the presence of the ALK4/ALK5/ALK7 inhibitor had no significant apoptosis-inducing effect  or non-manipulated Mino cells were cultured with or without TGF-β or BMP-7 for 3 days before detection of apoptotic cells. This approach demonstrated that the presence of truncated ALK5 almost completely counteracted BMP-7- and TGF-β-induced apoptosis  [The expression pattern of BMPs has not been fully explored in secondary lymphoid structures. However, our finding that BMP-7 is present in tonsillar B cells demonstrates that it is physiological relevant to study BMP-7-induced effects in B cells undergoing maturation and selection in the GC. We and others have demonstrated expression of oma (FL)  and mantoma (FL) . FL and BMP-7 has been shown to induce apoptosis in naïve and memory B cells from peripheral blood , as wellALK5 mutants, this receptor has been indicated to mediate TGF-β-induced apoptosis [Our data demonstrated that the apoptosis-inducing effect of BMP-7 could be counteracted by overexpression of truncated ALK5, but not by truncated ALK4 in Mino cells, suggesting that BMP-7 mediates its effects via ALK5. However, we were not able to show binding between BMP-7 and ALK5 (not shown), in line with previous data demonstrating that BMP-7 could bind to ALK2 and ALK3, but not to ALK5 , 47. Baspoptosis , 50. In poptosis . Therefopoptosis . Furtherpoptosis . Togethe+CD27hi plasmablast as demonstrated by us and others [BMP4 mRNA. Hence, HK cells could potentially influence the co-culture of GC B cells by production of BMPs and by competing for exogenously added BMPs. However, experiments with memory B cells cultured with or without HK cells did not indicate that HK cells interfered with BMP-induced effects.In the present study, we show that the combination of CD40L and IL-21 potently induced differentiation of GC B cells to CD38d others , 34. Thed others , 54. Thed others , 38, 55.d others . In addiIn conclusion, our work identified BMP-7 as a negative regulator of GC B-cell survival, hence adding another component to the regulation of B cells undergoing GC reaction. Our data suggests that BMP-7-induced apoptosis could be influenced by the TGF-β receptor ALK5 in an indirect manner, further demonstrating the receptor complexity within the TGF-β superfamily.S1 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.


Caenorhabditis elegans sperm are very efficient at navigating the hermaphrodite reproductive tract and locating oocytes. Here, we identify chemosensory and oxygen-sensing circuits that affect the sperm’s navigational capacity. Multiple Serpentine Receptor B (SRB) chemosensory receptors regulate Gα pathways in gustatory sensory neurons that extend cilia through the male nose. SRB signaling is necessary and sufficient in these sensory neurons to influence sperm motility parameters. The neuropeptide Y pathway acts together with SRB-13 to antagonize negative effects of the GCY-35 hyperoxia sensor on spermatogenesis. SRB chemoreceptors are not essential for sperm navigation under low oxygen conditions that C. elegans prefers. In ambient oxygen environments, SRB-13 signaling impacts gene expression during spermatogenesis and the sperm’s mitochondria, thereby increasing migration velocity and inhibiting reversals within the hermaphrodite uterus. The SRB-13 transcriptome is highly enriched in genes implicated in pathogen defense, many of which are expressed in diverse tissues. We show that the critical time period for SRB-13 signaling is prior to spermatocyte differentiation. Our results support the model that young C. elegans males sense external environment and oxygen tension, triggering long-lasting downstream signaling events with effects on the sperm’s mitochondria and navigational capacity. Environmental exposures early in male life may alter sperm function and fertility.The sperm’s crucial function is to locate and fuse with a mature oocyte. Under laboratory conditions,  C. elegans is a powerful genetic model to investigate the relationship between environment and male fertility. The hermaphrodite’s transparent epidermis permits the direct visualization of migrating male sperm and fertilization. In this study, we identified multiple serpentine receptor B (SRB) chemosensory receptors that are expressed in amphid sensory neurons, which extend cilia through the male nose. These SRB chemoreceptors are necessary to produce sperm that are efficient at navigating the hermaphrodite reproductive tract to the fertilization site. We show that SRB-13 signaling counteracts the negative effect of GCY-35 O2 sensor activity, thereby maintaining sperm mitochondrial function and navigational capacity in hyperoxic conditions. Of particular interest, SRB-13 acts in early larval stage males prior to testis maturation. We propose that young males respond to specific stressful environments by altering SRB neural circuits, which in turn impact sperm mitochondrial function and motility. This chemosensory mechanism may be part of a systemic response in C. elegans males to external environment and oxygen levels.Habitat loss, disease, climate change, and pollution are thought to negatively affect animal fertility. Sperm are a potential target, but the molecular mechanisms are not understood. The nematode  Animals employ sexual reproduction to increase genetic diversity critical for adapting to changing environments –3. An es2+ for movement . Th. Thsrb-1ractions  55]. Th. Thsrb-1 dataset , approxi feature . They al feature . These dsrb-13(xm1) males. The C. elegans mitochondrial genome encodes 12 protein complex subunits, 2 ribosomal RNAs, and 22 transfer RNAs (srb-13(xm1) mutant spermatids  mutation, which mildly reduces complex I function  males for 16 hours, and then to non-green fog-2(q71) control, srb-13(xm1);fog-2(q71), or srb-13,12,16(xmDf2);fog-2(q71) males for 16 hours. Mated females were separated from males and all green and non-green progeny were counted for a 24-hour period. (D) The percentage of progeny sired by the second (non-green) male. Bars, median value. *, p<0.005 using two-tailed Mann-Whitney U test. Additional underlying data can be found in (A) Cross sectional area measured from isolated male spermatids. Mean ± standard deviation. To the right is the number of spermatids measured. (B) Relative sperm number inseminated from (TIF)Click here for additional data file.S4 Figsrb-13 knock-in scheme using Mos1 transposon removal. The resulting allele is named xm4 and targeting plasmid is named pXM4. Homologous arms are indicated by dashed boxes. The locus is shown to scale, but primers are not. The DNA double-strand break site is indicated by a red line. A recombination event replaced srb-13 stop codon and 3’UTR by tdTomato tag, srb-13 stop codon and 3’UTR, and unc-119 rescue fragment. P1-4 and plasmid construction primers are listed in srb-16 knock-in scheme using Mos1 transposon removal. The resulting allele is named xm10 and targeting plasmid is named pXM10. Homologous arms are indicated by dashed boxes. The locus is shown to scale, but primers are not. The DNA double-strand break site is indicated by a red line. A recombination event replaced srb-16 stop codon and 3’UTR by tdTomato tag, srb-16 stop codon and 3’UTR, and unc-119 rescue fragment. P1-4 and plasmid construction primers are listed in spe-9 knock-in scheme using Mos1 transposon removal. The resulted allele is named xm14 and targeting plasmid is named pXM14. Homologous arms are indicated by dashed boxes. The locus is shown to scale, but primers are not. The DNA double-strand break site is indicated by a red line. A recombination event replaced spe-9 stop codon and 3’UTR by tdTomato tag, srb-16 stop codon and 3’UTR, and unc-119 rescue fragment. Note that srb-16 3’ UTR was used for cloning convenience. P1-4 and plasmid construction primers are listed in srb-13tdTomatoKI(xm4) PCR validation. The knock-in mutant was crossed into the fog-2(q71) background to generate males. PCR using P1-2 or P3-4 primers should amplify a 2.5kb or 2.6kb fragment, respectively, from the targeted locus only. NTC, no template control. (E) srb-16tdTomatoKI(xm10) PCR validation. The knock-in mutant was crossed into the fog-2(q71) background to generate males. PCR using P1-2 or P3-4 primers should amplify a 2.5kb or 2.6kb fragment, respectively, from the targeted locus only. (F) spe-9tdTomatoKI(xm14) PCR validation. The knock-in mutant was crossed into the fog-2(q71) background to generate males. PCR using P1-2 or P3-4 primers should amplify a 2.3kb or 2.5kb fragment, respectively, from the targeted locus only. (G) Wild-type N2 hermaphrodite uteri one hour after mating to indicated srb knock-in males. The spermathecae are outlined in yellow. Bar, 20 μm. Zone 3 sperm distribution value (mean % ± SEM) is shown. N, Number of scored uteri. All males are in fog-2(q71) background. Knock-in (KI) sperm are red due to MitoTracker labeling.(A) (TIF)Click here for additional data file.S5 Figosm-6p::dyf-11::GFP transgene (pO6D11FGP vector) marks sensory cilia (arrowheads). All strains were in the fog-2(q71) background. Bars, 20 μm (left panels) and 5 μm (magnified insets). (C) The osm-6 promoter (pOS13 vector) drives srb-13::mCherry expression specifically in male sensory neurons in the head and tail. Arrowheads indicate sensory neuron cell bodies. Arrows indicate sensory neuron dendrites. Transgenic osm-6p::srb-13mCherry lines show SRB-13::mCherry expression in sensory neuron cilia, throughout dendrites, and throughout cell bodies, likely due to overexpression. This contrasts with the SRB-13::tdTomato knock-in, which shows expression in sensory cilia, putative periciliary membrane compartment (myo-3 promoter (pMS13 vector) drives expression specifically in male body wall muscle, which is located in subdorsal and subventral quadrants. Bar, 100 μm. SRB-13::tdTomato and SRB-16::tdTomato knock-in expression in the adult male nose. The partment , and a f(TIF)Click here for additional data file.S6 Figsrb mutant and control male mitochondrial genomes. All males are in fog-2(q71) background. Mean ± S.E.M. Two-tailed Student’s t-test. *, p<0.005. (C) Mitochondrial genome showing selected RNAs and uaDf5 deletion (red). White arrows show positions of primers used in panel D. (D) PCR genotyping of control and uaDf5 worms showing heteroplasmy in uaDf5 males. (E) Mitochondrial genome RNA levels from uaDf5 mutant males relative to control males. Mean ± S.E.M. Two-tailed Student’s t-test. *, p<0.05; **, p<0.005; ***, p<0.0005. Additional underlying data can be found in (A) Principal component analysis, a statistical method to visualize variation and patterns in a dataset, of the six independent RNA-seq male datasets. Clustering of replicates is an indicator of reproducibility. (B) Raw RNA levels of selected mitochondrial genes from (TIF)Click here for additional data file.S7 Figfog-2(q71) in the N2 Bristol background. Bar, 20 μm. (B) Quantification of sperm distribution values (mean ± SEM). Number of scored uteri is on the right. *, p<0.0005 compared to the N2 x N2 zone 3 distribution. All wild isolates except for N2 aggregate on nematode growth plates seeded with E. coli. N2 is thought to have accumulated multiple mutations in O2-sensing circuits during laboratory cultivation, prior to initial cryopreservation. Only three isolates exhibit poor sperm performance under these conditions. These isolates, CB4856, DL238, and QX1211, exhibit high sequence divergence compared to the other isolates, which share large genomic regions in common. Additional underlying data can be found in (A) Hermaphrodite uteri images one hour after mating to indicated males. Strains used are shown in the upper left. Fluorescent sperm are red due to MitoTracker labeling. Spermathecae are outlined in yellow. Control males are (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Navigator gating is commonly used to minimize respiratory motion in free-breathing whole-heart coronary MRA [1]. However, lengthy and unpredictable acquisition times remain a drawback. Respiratory self-navigation (SN) [2-3], conversely, enables 100% scan efficiency, but performs motion correction over a broad range of respiratory displacements, which can result in image artifacts. Here, we propose an alternative respiratory motion-resolved approach based on 3D radial phyllotaxis sampling, respiratory motion sorting and sparse reconstruction.3, matrix 1923, voxel (1.15 mm)3, RF angle 115°, and receiver BW 898 Hz/Px. Using a respiratory signal directly extracted from the modulations of the k-space center amplitude within the radial imaging data [5], signal-readouts were grouped according to the respiratory state at which they were acquired  were performed on a 1.5T clinical MRI scanner  with a prototype 3D radial phyllotaxis bSSFP sequence [4]: TR/TE 3.1/1.56 ms, FOV (220 mm)Respiratory-resolved XD-GRASP reconstruction effectively suppresses respiratory motion artifacts Fig. . AverageInstead of discarding data or enforcing motion models for motion correction, XD-GRASP makes constructive use of all respiratory phases to improve image quality, and achieves superior quality compared to 1D respiratory SN without the need for breath-holding, navigators, or complex 3D respiratory motion correction schemes. The phyllotaxis trajectory and XD-GRASP reconstruction provide a synergistic combination that may lead routine coronary MRA closer to clinical practice.


The mechanistic target of rapamycin (mTOR) is a major regulator of cell growth and is frequently dysregulated in cancer. While mTOR complex-1 (mTORC1) is a validated cancer target, the role of mTOR complex-2 (mTORC2) remains less defined. Here, we reveal mTORC2 as a critical regulator of breast cancer metabolism. We showed that hyperphosphorylation in ATP citrate lyase (ACL) occurs frequently in human breast tumors and correlates well with HER2+ and/or PIK3CA-mutant (HER2+/PIK3CAmut) status in breast tumor cell lines. In HER2+/PIK3CAmut cells, mTORC2 controls Ser-455 phosphorylation of ACL thereby promoting acetyl-CoA production, de novo lipogenesis and mitochondrial physiology, all of which were inhibited by an mTORC1/mTORC2 kinase inhibitor (mTOR-KI) or cellular depletion of mTORC2 or ACL. mTOR-KI but not rapamycin blocked the IGF-1-induced ACL phosphorylation and glucose to lipid conversion. Depletion of mTORC2 but not mTORC1 specifically inhibited the ACL-dependent acetyl-CoA production. In the HER2+/PIK3CAmut MDA361, MDA453, BT-474 and T47D cells, depletion of mTORC2 or ACL led to growth inhibition and mitochondrial hyperpolarization, which were partially rescued by an alternate source of acetyl-CoA. These same changes were not apparent in mTORC2- or ACL-depleted HER2-/PIK3CAwt MDA231 and HCC1806 cells, highlighting a differential dependence of mTORC2-ACL for survival in these two cell types. Moreover, ACL Ser-455 mutants S455E (phosphomimetic) and S455A (non-phosphorylatable) each increased or decreased, respectively, the acetyl-CoA production, mitochondrial homeostasis and survival in ACL-depleted MDA453 cells. These studies define a new and rapamycin-resistant mechanism of mTORC2-ACL in lipogenesis and acetyl-CoA biology and provide a rationale for targeting of mTORC1 and mTORC2 in HER2+/PIK3CAmut breast cancer. The mechanistic target of rapamycin (mTOR) acts through two multiprotein complexes, mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2), coordinates signals from growth factors, nutrients and energetics. mTOR functions both in parallel and downstream of the PI3K/AKT signaling pathway that is frequently dysregulated in cancer and a subject of intense discovery research . While cmTOR plays an important role in pathogenesis and therapy resistance in breast cancer , 17. As Cancer cells feature aerobic glycolysis because it not only satisfies energy production but also generates cellular building-blocks including those for de novo lipid biosynthesis , 22. TheWe sought to investigate novel role of mTOR in glucose to lipid metabolism in breast cancer, particularly those governed by mTORC2, a key component of HER2, PI3K pathway and is resistant to rapalog therapy. We performed phosphoproteomic analysis of MDA361, a HER2+/PIK3CAmut breast tumor line, treated without or with a highly selective mTOR-KI WYE-125132 (WYE-132) . Herein,versus the control “light”-labeled phosphopeptides (H/L) for Ser-455-containing peptide was 0.24, indicating a 76% inhibition due to WYE-132 treatment , hyperplasia (n = 10), invasive ductal carcinoma (n = 18) and invasive lobular carcinoma (n = 8) revealed a general trend for low staining in normal breast, a noticeably increased staining in hyperplasia and the highest P-ACL staining in invasive ductal and lobular carcinoma  and P-AKT(S473) profile in a panel of breast cancer cell lines. In a panel of 10 breast tumor lines, 6 cell lines  exhibited elevated and constitutive levels of P-ACL, while 3 cell lines  expressed low P-ACL levels and 1 line (MCF7) showed medium level of P-ACL that was repressed upon serum withdrawal  on normal- and breast tumor tissues. We first validated the antibody specificity in cultured cells treated with DMSO or WYE-132, in which a positive staining was greatly diminished upon WYE-132 treatment . P-ACL Ia Figure , left. Ta Figure , right. l Figure . The levl Figure . In thisl Figure . Collectvia apoptosis, whereas similar treatment of HER2-/PIK3CAwt cells (HCC1806 and MDA231) inhibited growth without cell death  with 2 μmol/L of two independent mTOR-KIs, WYE-132 or AZD8055, each caused a substantial cell death h Figure . This seh Figure .p < 0.001) without affecting total ACL, while similar depletion of mTORC2 in HER2-/PIK3CAwt MDA231 or HCC1806 cells did not reduce P-ACL   in MDA453 cells  or ACL-depleted (p < 0.01) and, as expected, in raptor-depleted (p < 0.05) MDA453 cells  , 28. Giv) Figure , thus fus Figure . Likewiss Figure . Notablys Figure , indicatp < 0.01)     Figure . For com) Figure , which i) Figure . While b) Figure , and its) Figure . The resvia supplementing sodium acetate (NaAc) to culture medium , while the glutamic acid mutant ShR-SE further increased acetyl-CoA relative to that of ShR-WT (p < 0.05)   Figure . To asse) Figure . In pcDN) Figure . We alsos Figure . The fres Figure . These rversus HER2-/PIK3CAwt cells   and by the HER2 kinase inhibitor Lapatinib (p < 0.01)  Figure . Consist) Figure . Lastly,) Figure . CollectSuccessful development of mTOR-targeted cancer treatment will require molecular understanding of mTORC1 and mTORC2 in oncogenesis and therapy resistance. While mTORC1 has been a validated cancer drug target for years, the role of mTORC2 remains much less defined. More recent studies have suggested an essential role of mTORC2 in cellular transformation driven by oncogenic HER2  or EGFRvin vitro. While this observation is interesting, it is unknown whether mTORC2 directly phosphorylates ACL in vivo. The observation that both WYE-132 and MK-2066 inhibited phospho-ACL is consistent with the notion that mTORC2 signals, at least in part, through AKT to phosphorylate ACL  glucose . Total lipids were extracted twice with 300 μL each of hexane:isopropanol  as described [escribed . Cell lyDetailed method for MDA361 cell SILAC study has been described previously .


Scientific Reports 10.1038/s41598-017-12234-5, published online 18 September 2017Correction to: This Article contains errors within Figure 7, in which certain curves are distorted. The correct Figure 7 appears below as Figure


Calcineurin (Cn) is a calcium activated protein phosphatase involved in many aspects of normal T cell physiology, however the role of Cn and/or its downstream targets in leukemogenesis are still ill-defined. In order to identify putative downstream targets/effectors involved in the pro-oncogenic activity of Cn in T-cell acute lymphoblastic leukemia (T-ALL) we used tandem affinity chromatography, followed by mass spectrometry to purify novel Cn-interacting partners. We found the Cn-interacting proteins to be part of numerous cellular signaling pathways including eIF2 signaling and mTOR signaling. Coherently, modulation of Cn activity in T-ALL cells determined alterations in the phosphorylation status of key molecules implicated in protein translation such as eIF-2α and ribosomal protein S6. Joint targeting of PI3K-mTOR, eIF-2α and 14-3-3 signaling pathways with Cn unveiled novel synergistic pro-apoptotic drug combinations. Further analysis disclosed that the synergistic interaction between PI3K-mTOR and Cn inhibitors was prevalently due to AKT inhibition. Finally, we showed that the synergistic pro-apoptotic response determined by jointly targeting AKT and Cn pathways was linked to down-modulation of key anti-apoptotic proteins including Mcl-1, Claspin and XIAP. In conclusion, we identify AKT inhibition as a novel promising drug combination to potentiate the pro-apoptotic effects of Cn inhibitors. T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 15% of pediatric and 25% of adult cases of ALL. Progress in T-ALL therapy has been impressive, with cure rates approaching 80% for children and 50% for adults -5.Calcineurin  is a calcium-activated serine/threonine (S/T) phosphatase, composed of a catalytic subunit (PPP3CA) and a regulatory subunit (PPP3CB), critical to a number of developmental processes in the cardiovascular, nervous, and immune system. Cn has the ability to dephosphorylate a broad range of proteins, including Nuclear Factor of Activated T cells (NFAT) proteins. Although critically involved in many aspects of normal T cell survival, proliferation and activation, the direct implication of Cn and/or its downstream NFAT targets in lymphomagenesis and cancer in general has only recently been reported , 7. AvaiTo identify novel factors that support the putative oncogenic function of Cn in T-ALL, we purified activated PPP3CA from Jurkat T-ALL cells. To this end, we generated Jurkat cells stably expressing a human constitutively active PPP3CA mutant (PPP3CA*) tagged with both FLAG and HA epitopes (F/H-PPP3CA*). This mutant lacks both the calmodulin binding domain and auto-inhibitory domains and is constitutively active  even in To validate our results, proteins belonging to the major DAVID functional annotation clusters identified and distributed relatively homogeneously in our ordered protein list  were cloTo identify signaling pathways enriched in the identified PPP3CA-binding partners we made use of the Ingenuity pathways analysis software . The top canonical signaling pathways are shown in Figure in vivo in mouse models of human T-ALL/lymphoma [Persistent Cn/NFAT signaling has been shown to be pro-oncogenic lymphoma  and verylymphoma . Since slymphoma  or FK-50lymphoma  to calcuIn 50-75% of T-ALL patients, PI3K/Akt/mTOR signaling pathway is constitutively active and negatively affects patient outcome . To try We also evaluated the effects of these drugs on activated peripheral blood mononuclear cells . PBMCs were isolated from normal donors and treated with different concentrations of the Cn inhibitors  or PI3K/AKT/mTOR pathway inhibitors BEZ235 and MK-2206, before being evaluated for apoptosis induction. We found that PBMCs remained highly viable even at very high concentrations . In addiTo assess which survival factors were responsible for the strong anti-leukemic effects of the combination treatment MK-2206 and CsA, we used antibody-based apoptosis arrays. To this end, Jurkat cells were treated for 24h with vehicle, MK-2206, CsA or the combination MK-2206 plus CsA. Cell lysates were then hybridized to human apoptosis arrays, which allow the simultaneous detection of 35 apoptosis-related proteins. In Jurkat cells, the combination treatment MK-2206 and CsA determined a strong induction of cleaved Caspase 3, which was associated with a strong reduction in numerous anti-apoptotic proteins including survivin, Claspin and Bcl-2 Figure . Some ofUsing an unbiased proteomic approach to identify proteins possibly relevant to the oncogenic properties of Cn in T-ALL, we identified novel and known PPP3CA-interacting proteins implicated in numerous cellular signaling pathways, including eIF2 signaling, cell cycle control, mTOR signaling and 14-3-3 mediated signaling. Importantly, several proteins implicated in leukemia pathobiology emerged amongst the novel PPP3CA interactors such as Rb, NPM1, BCL11b, GSK3β and KDM1 . Of these, GSK3β has been described to interact with Cn in neuron-derived cells  and receCalcium signaling  has been linked to numerous aspects of tumorigenesis, influencing motility, angiogenesis, genotoxicity, gene transcription, telomerase activity, differentiation, cell cycle progression and apoptosis . InteresMcl-1 mRNA translation [By inhibiting pathways enriched in our PPP3CA protein complex we identified the dual PI3K-mTOR inhibitor BEZ235 as a promising drug in synergizing with Cn inhibitors to promote T-ALL cell death. Further studies to dissect the key element/s of the PI3K/AKT/mTOR pathway whose inhibition synergize best with Cn inhibitors, identified AKT as the key component to inhibit. Mechanistically, we find that joint AKT and Cn inhibition promotes cytotoxicity in T-ALL cells through the down-regulation of key anti-apoptotic proteins including Mcl-1, Claspin and XIAP. Of these, Mcl-1 down-regulation emerged as the most preserved mechanism across T-ALL cell lines and xenografts. Mcl-1 is a critical mediator of cell survival, especially of hematopoietic cells , and itsnslation , 29 and nslation , 31.The PI3K/AKT as well as mTOR pathways are two interconnected signaling pathways affecting numerous cellular functions, including metabolism, growth and survival , 33. In MK-2206 is a novel, orally active, allosteric AKT inhibitor, which is under development for the treatment of solid tumors. MK-2206 is a potent and selective inhibitor for AKT, whose efficacy has been proven in preclinical models of human cancer , 35. In +/CD7−/CD4− T-ALL subset [It is believed that difficulty in eradicating tumors could result from conventional treatments targeting the bulk of tumor cells, but not the putative Cancer Stem Cells (CSCs) or LICs, thus strategies aimed at eliminating these cells could have significant clinical implications. MK-2206 has been shown to be able to markedly reduce the CD34L subset , which hL subset . Thus, iIn conclusion, our study has uncovered novel signaling pathways which interact with the Cn-NFAT signaling pathway in T-ALL cells including eIF2 signaling and p70 S6K/mTOR signaling. Interestingly, we demonstrate an enhanced anti-leukemic effect of combination strategies involving joint inhibition of AKT with Cn inhibition. Hopefully, the identification of novel interactions between canonical pathways deregulated in T-ALL for which clinically relevant inhibitors are available (such as Cn-NFAT and PI3K/AKT/mTOR pathways) could lead to to a more effective therapy of T-ALL patients still facing a poor prognosis.9 Jurkat cells stably expressing FLAG-HA tagged PPP3CA* constitutively active mutant (F/H PPP3CA*) and control Jurkat cells were harvested by centrifugation, washed in cold PBS and resuspended in CO-IP lysis buffer  supplemented with phosphatase and protease inhibitor cocktails. The lysate was cleared by centrifugation at 13.000 g for 30 min. Whole cell extracts were incubated for 16 h (at 4°C with rotation) with anti-FLAG M2 agarose beads  (1% v/v) equilibrated in BC100 . Beads were washed 3 times with BC100 containing 0.3% Triton X-100, and bound proteins were eluted with 4 bead volumes of BC100 containing 0.5 mg/mL of FLAG peptide (Sigma-Aldrich) for 6 h. The FLAG affinity purified complexes were further immunopurified by using 30 μL of anti-HA conjugated beads (Sigma-Aldrich). After incubation for 16 h, HA beads were washed 4 times with BC-100 containing 0.2% Triton X-100 in spin columns  and eluted under native conditions using HA peptide . Ten percent of the eluate was resolved on SDS-PAGE and silver stained using the silverquest kit . The remaining material was TCA precipitated and subsequently analysed by MS at the Taplin Biological Mass Spectrometry facility . After compiling and filtering the data , the protein list was queried on the DAVID functional annotation clustering platform to identify protein complex functions [For the purification of Cn-associated proteins, 12 × 10unctions . With meunctions . The filBEZ235, CAL-101, GSK650394, Roscovitine, Salubrinal, AZD8055, MK-2206 and PF4708671 were from Selleck Chemicals LLC . CN585 -4--2-phenyl-pyrimidine) was from Merck Millipore . Ionomycin, PMA, CsA, FK-506 and BV-02 were from Sigma-Aldrich.2, 0.1% NP-40, 10 mM Imidazole, 10% Glycerol) supplemented with protease inhibitors. Cell lysates were incubated with HisPur Cobalt resin (Pierce) overnight at 4°C. Beads were washed three times with lysis buffer and proteins were eluted by incubating the beads with 300 mM Imidazole elution buffer. Immune complexes were analyzed by SDS-PAGE and WB. For IP of endogenous PPP3CA bound proteins in T-ALL cells, we lysed 100-150×106 T-ALL cells for 30 min in RIPA lysis buffer supplemented with phosphatase and protease inhibitor cocktails. After centrifugation, the cell lysates (diluted 1:3 with CO-IP buffer) were pre-cleared with TrueBlot® anti-Mouse Ig IP Beads  before being incubated overnight with 5 μg of mouse antibody against PPP3CA  or irrelevant mouse Ig . Subsequently, samples were incubated for 2h with TrueBlot® anti-Mouse Ig IP Beads and IP washed 5 times with CO-IP buffer. Immune complexes were then analyzed by SDS-PAGE and immunoblot.Human embryonic kidney (HEK) 293T cells were transfected with expression vectors encoding pcDNA3-F/H-PPP3CA* and each of the expression vectors encoding the novel PPP3CA interacting partners  using JetPEI transfection reagent . Fourty hours after transfection, cells were lysed in CO-IP buffer . For IP, cell lysates were incubated with anti-HA or anti-FLAG M2 affinity gel beads (Sigma-Aldrich) overnight at 4°C. Beads were washed five times with lysis buffer and proteins were eluted by incubating the beads with HA peptide  or FLAG peptide  or boiled in 5X SDS-Laemmli's sample buffer. Immune complexes were analyzed by SDS-PAGE and immunoblot. For His-tagged proteins, transfected cells were lysed in TEN buffer  immunodeficient mice. Primary T-ALL cells from xenografted mice were cultured in vitro in MEM-alpha media supplemented with 10% human serum and cytokines [6/mL per well in complete medium and stimulated with a mixture of anti-CD3  and 50 ng/mL human recombinant IL-2  to induce proliferation. Drugs were added to PBMCs for 48 h before evaluation of apoptosis induction.HEK 293T were maintained in DMEM containing 10% fetal bovine serum (FBS) and 0.05 mg/ml penicillin/streptomycin. T-ALL cell lines were maintained in RPMI-1640 media supplemented with 10% FBS and 0.05mg/ml penicillin/streptomycin (complete medium). Primary xenograft T-ALL cells were expanded Total cell lysates were prepared using RIPA lysis buffer supplemented with phosphatase inhibitor cocktail set I and II (Sigma-Aldrich) and protease inhibitor cocktail tablets (Roche) and normalized for protein concentration using the BCA method (Pierce). For Western blotting, protein samples were separated on 4-12% gradient Tris-Glycine or 3-8% Tris-Acetate SDS-PAGE (Invitrogen) and transferred to PVDF membrane . Antibodies against c-myc (9E10), NFATc2 and Claspin were from Santa Cruz Biotechnology; antibodies recognizing GSK3β, PPP3CA, β-actin, MCM-4, FLAG epitope, GST, phospho-mTOR (S2448), mTOR, phospho-AKT (S473), AKT, phospho-eIF2α (S51), eIF2α, phospho-eIF4E (S209), phospho-p70 S6K (T389), XIAP, Mcl-1, survivin, Claspin, Bim, phospho-S6RP (S235/236) and p70 S6K, were from Cell Signaling Technologies ; HA epitope antibody was from Roche; mouse anti-human Rb protein (BD Pharmingen), rabbit anti-BCL11b ; mouse FLAG epitope antibody (M2), NFATc2 antibody (HPA 008789) were from Sigma-Aldrich; V5 tag antibody (E10/V4RR) was from Thermo Scientific  and Green Fluorescent Protein antibody (A11122) was from Invitrogen.We analysed the expression profiles of 35 apoptosis-related proteins on cellular extracts using the Human Apoptosis Array . Approximately 150 μg of cell lysates were incubated overnight with the Human Apoptosis Array. The BioRad ChemiDoc XRS Imager was used to capture the signals from the arrays. The density of each spot was quantified by Quantity One software (BioRad). Raw signal intensities in each array were taken average across duplicate spots for each probe and negative control probe (PBS) was subtracted. Then processed data were expressed relative to DMSO control and heat map generated using GenePattern .FLAG tagged PPP3CA , constitutively active FLAG-tagged PPP3CA mutant lacking the calmodulin binding and autoinhibitory domains , double tagged FLAG/HA PPP3CA* mutant (F/H-PPP3CA*), KIF-11, PRMT5, BCL11b and NPM1 expression vectors (in pcDNA3 backbone) were generated by standard cloning procedures. Expression constructs for RBBP4, hnRNPU, KHSRP, IFI-16, FLNa, RPA2, PKM2, FUS, ARG1, STK38, PRDX1, DNAJA2, GSK3β and HSPA8 were from Addgene.The retroviral vector (MigR1tomato-F/H PPP3CA*), expressing the red fluorescent protein Tomato and F/H-PPP3CA* was generated by subcloning F/H-PPP3CA* from pcDNA3-F/H-PPP3CA* into the MigR1tomato vector using BglII and XhoI restriction sites.Retroviral particles were generated according to standard protocols.Cell viability in T-ALL cell lines was analyzed via the bioluminescent method Vialight plus  after 72 h. This assay allows bioluminescent detection of cellular ATP as a measure of viability. Measurement of ATP is the most accurate, effective, and direct way of determining the number of living cells in culture. We also analyzed apoptosis after 48-72 h by flow cytometry (FACS) after staining with Annexin V-FITC (Roche) and SYTOX Red dead cell stain (Invitrogen). The samples were collected on a FACSCalibur  using Cell Quest software (BD Biosciences), and analysed with FlowJo (Tree Star).t-test and Mann-Whitney U test. All statistical tests were two sided and P<0.05 was considered statistically significant. We analyzed drug synergism using the median-effect method of Chou and Talay [Statistical analysis was performed by Student's nd Talay  and used


Finally, rescue experiments with a lethal mfap1 mutant show that the VCP binding region is not essential for Mfap1 function, but may act to increase its stability or activity.Posttranscriptional regulation of gene expression contributes to many developmental transitions. Previously, we found that the AAA chaperone Valosin-Containing Protein (VCP) regulates ecdysone-dependent dendrite pruning of Drosophila class IV dendritic arborization (c4da) neurons via an effect on RNA metabolism. In a search for RNA binding proteins associated with VCP, we identified the spliceosome-associated protein Mfap1, a component of the tri-snRNP complex. Mfap1 is a nucleolar protein in neurons and its levels are regulated by VCP. Mfap1 binds to VCP and TDP-43, a disease-associated RNA-binding protein. via distinct regions in its N- and C-terminal halfs. Similar to  Drosophila, pruning occurs primarily during metamorphosis, when the larval nervous system develops into the adult one. For example, the peripheral sensory class IV dendritic arborization (c4da) neurons prune their extensive larval dendritic arbors during the first sixteen hours after puparium formation (h APF) [Sox14, Mical, and headcase [VCP, also known as TER94), cause defects in dendrite pruning and ecdysone-induced neuronal apoptosis [Mical, likely through induction of a different Mical splice isoform [Pruning, the regulated loss of synapses or neurites during neuronal development, is an important specification mechanism that contributes to the mature morphology of neurons . In Dros (h APF) ,3. C4da  (h APF) ,3. Ecdysheadcase . These gheadcase ,7 In addheadcase . We prevpoptosis ,9. VCP ipoptosis ,11. Thespoptosis ,11. Duri isoform . In addi isoform .Caenorhabditis elegans, mfap-1 mutations cause alterations in alternative splicing of a reporter gene, with both intron retention and exon skipping [mfap1 mutant. Rescue experiments show that the N-terminal 229 amino acids of Mfap1 containing the VCP binding site are not required for viability, but confer overexpression toxicity in te context of full length Mfap1. Thus, VCP binding may serve to stabilize the spliceosome-associated protein Mfap1.Based on these observations, we hypothesized that VCP regulates dendrite pruning via an effect on mRNA metabolism. In a search for splicing factors interacting with VCP, we identified the spliceosome-associated Microfibril-associated protein 1 (Mfap1). Mfap1 binds to Prp38, a component of the tri-small nuclear ribonucleoprotein (tri-snRNP) complex, an intermediate complex during the splicing reaction, and is required for splicing of a cell cycle-related mRNA . In Caenskipping . The C-tskipping . In thisDrosophila Mfap1, a spliceosome-associated protein. Control c4da neurons have long and branched dendrites at the third instar larval stage  . Under tThe Cajal body marker coilin-YFP  also locAs Mfap1 staining was enhanced by expression of the dominant-negative VCP QQ, we tested whether Mfap1 and VCP can form a complex. To this end, we co-transfected C-terminally GFP-tagged Mfap1 and FLAG-tagged VCP in S2 cells and performed co-immunoprecipitation experiments. We could not detect wild-type VCP in Mfap1-GFP immunopreciptates , however1-229 is 4.6). VCP QQ selectively and strongly bound to the N-terminal GFP-Mfap11-229, but not the C-terminal GFP-Mfap1230-478 fragment . Since mfap1DsRed is lethal prior to the pupal phase, we could only assess the effect of mfap1DsRed heterozygousity. However, also this manipulation did not significantly suppress the pruning defects induced by VCP QQ ED4287, a deficiency uncovering the MFAP1 locus , the N-terminal fragment encompassing the VCP binding site (UAS-GFP::Mfap11-229), and the conserved C-terminal fragment (UAS-GFP::Mfap1230-478). We expressed these under the control of the ubiquitous GAL4 driver actin-GAL4 and asked which of them could rescue the lethality of transheterozygous mfap1DsRed/Df(3L)ED4287 flies. At 25°C, the full-length Mfap1 construct rescued the lethality, but only in the absence of the GAL4 driver, i. e., through leak expression from the UAS promotor  rescued the lethality and gave viable flies both in the absence and presence of actin-GAL4 , UAS-Mfap1 RNAi (VDRC 15610), UAS-coilin::YFP , ppk-GALppk-GAL4 , UAS-tdtppk-GAL4 , UAS-VCPppk-GAL4 , UAS-mchMFAP1 stop codon were designed with the CRISPR Optimal Target Finder (http://tools.flycrispr.molbio.wisc.edu/targetFinder/index.php) and cloned into pCFD3. Forward gRNA sequences were:gRNAs corresponding to sites in the 5’UTR and 100 bp in front of the gtcgAAAGATTTGCTTATTACCCG,gRNA1: gtcgCTGGATGACTCGGCGTACCA.gRNA2: Mfap1 homology arms (ca. 1.5 kb outside of the respective 5’ and 3’ gRNAs) were cloned into pBluescript such that they flanked the 3P3-DsRed marker. Homology arm primers were:CGTCTCAggacGTTTAATTGCTGGTTCTTGG (fw), CGTCTCActggGTAATAAGCAAATCTTTGTATTTATTCC (rev);left arm, CGTCTCAtgttTACGCCGAGTCATCCAGCAA (fw), CGTCTCAgcatAGGCAACTGTGGAAAAGCCT (rev).right arm, act-Cas9, lig4-/- flies [Df(3L)ED4287 (Bloomington 8096) uncovering the MFAP1 locus, and characterized by PCR and sequencing. A line, designated mfap1DsRed, was recovered that had the cassette inserted at the 3’ CRISPR target approximately 100 base pairs in front of the MFAP1 stop codon. Primers for genomic PCR were:The gRNA plasmids and the homology cassette were injected into /- flies . InjectaTCCACGAATGGCCGGAAAAT and XZ85 [upstream PCR: and XZ85 ,CTACAGGAGCACACGCTCAA.downstream PCR: XZ120 , CTACAGG1-229 and Mfap1230-478 were cloned into pUAST attB GFP and inserted into the second chromosome 44F landing site [HATDP-43 Δ331–388 (Δgly) was generated by PCR from pUAST attB HATDP-43 [Sequences corresponding to full length Mfap1 1–478), Mfap1, Mfap11-Live images of larval or pupal c4da neurons at 18 h APF were taken on a Zeiss LSM710 confocal microscope, and dendrite and pruning phenotypes were quantified as previously described .Antibody stainings of larval filets were done as decribed  except tThe indicated UAS plasmids were cotransfected with actC5-GAL4 into S2 cells and allowed to express for 72 hours. Lysis and immunoprecipitation were performed according to standard methods . For immS1 Figppk-GAL4  were stained for Mfap1 at the third instar larval stage. Panels (A), (B) show Mfap1 stainings, panels (A’), (B’) show the merge with GFP to depict the c4da neuron soma. The soma is also indicated by a dashed line in panels (A) and (B). The arrow in (A) depicts the characteristic Mfap1 dot.Control c4da neurons  or c4da neurons expressing Mfap1 RNAi under (EPS)Click here for additional data file.S2 FigCotransfected UAS constructs are indicated on top. A FLAG blot for FLAG-tagged VCP versions. Lanes 1–4 were shown in (EPS)Click here for additional data file.S3 FigCotransfected UAS constructs are indicated on top. A FLAG blot for FLAG-tagged VCP QQ. Lanes 1–4 represent input samples shown in (EPS)Click here for additional data file.S4 FigCotransfected UAS constructs are indicated on top. A HA blot for HA-tagged TDP-43 versions. Lanes 1–4 were shown in (EPS)Click here for additional data file.S5 FigCotransfected UAS constructs are indicated on top. A HA blot for HA-tagged TDP-43 versions. Lanes 5–8 are shown in (EPS)Click here for additional data file.S6 FigActin-Cas9) or mfap1DsRed/Tm6b mutant flies. A Genomic PCR using primers for the Mfap1 upstream region and the DsRed cassette. The expected 3.2 kb band is only seen in the mutant. B Genomic PCR using primers for the Mfap1 downstream region and the DsRed cassette. The expected 0.9 kb band is only seen in the mutant.PCRs were done on genomic DNA from control flies ((EPS)Click here for additional data file.


Scientific Reports 10.1038/s41598-017-07404-4, published online 22 September 2017Correction to: This Article contains typographical errors in the Acknowledgements section.“Ruigrok was supported by a clinical fellowship grant of the Netherlands Organization for Scientific Research (NWO) (project no. 40-00703-98-13533).”should read:“Ruigrok was supported by a clinical fellowship grant of the Netherlands Organization for Scientific Research (NWO) (project no. 90714533).”


Toxorhynchites amboinensis (TaOr8) suggests a different biological role. Here, we show that TaOR8 is a functional ortholog of its counterparts in blood-feeding mosquitoes displaying selectivity towards the (R)-enantiomer of octenol and susceptibility to the insect repellent DEET. These findings suggest that while the function of OR8 has been maintained throughout mosquito evolution, the context in which this receptor is operating has diverged in blood and nectar-feeding mosquitoes.Olfaction is a key insect adaptation to a wide range of habitats. In the last thirty years, the detection of octenol by blood-feeding insects has been primarily understood in the context of animal host-seeking. The recent discovery of a conserved octenol receptor gene in the strictly nectar-feeding elephant mosquito  Ors) play a major roleThe molecular mechanisms by which insects detect odor cues involve many gene families of which odorant receptors (R)-(−)-1-octen-3-ol (thereafter termed (R)-octenol) is of particular interest for several reasons. First identified as a tsetse attractant released by cattle67592Anopheles and Aedes but in Culex, octenol elicits little to no attractive effectsThe survival of most mosquitoes depends on locating animal hosts, resting and oviposition sites, as well as suitable sources of nectar 2. The roLantana camara release (R)-octenolAe. aegypti exhibit octenol-sensitive basconic sensillaOr8 gene (AaOr8)Culex quinquefasciatus mosquitoes is unclear as multiple OR8 paralogs are activated by high doses of octenol13Ae. aegypti have been found in proximity to hosts, perhaps as a means to increase their likelihood to locate a mate, and that octenol has not been excluded as a bird emanation. In any case, the 145–200 million years old conservation of the octenol receptor OR8, in Culicinae and Anophelinae mosquitoes18Octenol may play a role in nectar-seeking as proposed by earlier authorsToxorhynchites might be valuable to explore the role of octenol since animal host-seeking is not part of their behavior23Toxorhynchites group, which belongs to the Culicinae subfamily, separated from the Aedes and Culex lineages about 38–54 million years ago22T. amboinensis belong to a small group of nectar-feeding mosquitoesOr homologs with Ae. aegypti including Or8 (TaOr8)T. amboinensis and Ae. aegypti express Or8 at similar levels in the maxillary palps suggesting a conserved key role in the life cycle of adult mosquitoes. Resolving the tuning properties of TaOR8 may clarify the role of this receptor outside animal host-seeking paradigms. Using a cell-based functional assay, we show that TaOR8’s response to octenol is highly sensitive, enantioselective, inhibited by the insect repellent DEET and odorant specific. Common ancestral origin and functional conservation support TaOR8 as a functional ortholog of the Ae. Aegypti, Anopheles gambiae and Culex quinquefasciatus OR8s. These features provide evidence that TaOR8 is an octenol receptor whose ecological role is unknown but excludes animal host-seeking. These findings question the ecological role traditionally ascribed to the OR8/(R)-octenol partner in blood-feeding mosquitoes and suggest that octenol may be useful to mosquitoes in multiple contexts beyond animal host-seeking.The recent discovery of the OR repertoire in the Elephant mosquitoes Aedes-Toxorhynchites split and different ecological requirements -(−)-1-octen-3-ol and (S)-(+)-1-octen-3-ol enantiomers (R) enantiomer is the predominant form1529Xenopus laevis oocytes and recorded the responses of this receptor complex to the (R), (S) and racemic mixture (RS) of 1-octen-3-ol using the two-microelectrode voltage clamp technique. An example of a current trace is shown in 50 values show that the (R) enantiomer (EC50: 401 nM) is approximately 8 and 126 times more potent than the (RS) mixture  and the (S) enantiomer , respectively. Although the (S) contained <0.1% or no (R) at all, we cannot exclude the possibility that TaOR8 response was elicited by trace amount of the (R) enantiomer. Sensitivity in the nanomolar range for (R)-octenol supports a cognate receptor ligand relationship, which is comparable to pheromone receptor-pheromone pairsOctenol is a chiral compound composed of the (ntiomers . The (R)−3 M) (R)-octenol (10−7 M), as carried out previously with AaOR8R)-octenol alone did not differ before and after exposure to the (R)-octenol-DEET mix, indicating no adaptive effect (−7 M (R)-octenol elicited identical TaOR8 responses, excluding a potential position effect.To further confirm that both mosquito receptors are functional orthologs, we tested the inhibitory effect of DEET (10−3 M)  on the Te effect . ApplyinR)-octenol) belonging to 10 classes of organic compounds  to explore the odor space of TaOR8 -octenol. (R)-octenol was the most potent ligand eliciting a response 30 times higher than the next most potent chemical 3-octanone, also an 8-carbon aliphatic compound, and 46 times higher than 1-hepten-3-ol, which is identical to octenol except for one carbon shorter.We used a panel of 29 compounds including -octenol, (iv) feature a susceptibility to DEET inhibition, (v) are narrowly tuned to (R)-octenol. Such evolutionarily conservation of receptor biochemical function in Toxorhynchites is surprising since this species does not animal host-seek23This study shows that TaOR8 and AaOR8 are functional orthologs as both (i) share a high level of sequence identity, (ii) are expressed in the maxillary palps, (iii) exhibit high sensitivity (nanomolar range) towards . Identifying other contexts in which this compound is used by mosquitoes will be challenging, as octenol is a common environmental volatile that may serve multiple roles in mosquito behavior. Octenol is synthesized by fungi35153738340Our results support a role of TaOR8 outside an animal host-seeking context. Since TaOR8 requires ORco, this is consistent with the discovery that Oryzaephilus surinamensisPhormia reginaMaruca vitrataLobesia botranaEupoecilia ambiguellaLutzomyia longipalpisMegaselia halterataLariophagus distinguendusOctenol is used as an aggregation pheromone in the sawtoothed grain beetle, Ae. aegyptiT. amboinensisBactrocera dorsalisDelia platuraIn Diptera, octenol has been suggested to act as an oviposition attractant. First mentioned as a potential oviposition cue for Ae. aegypti? The ecological context in which AaOR8 operates may be restricted to animal host-seeking, but perhaps more intriguing is the possibility that AaOR8 is involved in eliciting multiple behaviors. As a ubiquitous cue, octenol may be used by mosquitoes in combination with other cues, olfactory or otherwise2 and other volatiles may be used as an animal host attractantR)-octenol on blood-feeding mosquitoes will be necessary to reveal the entire ecological contexts of its biochemical function.What is the role of OR8 in Toxorhynchites amboinensis by trizol extraction. First strand cDNA synthesis was carried out using the TranscriptorTM kit , according to the manufacturers protocol. PCR amplification of full-length TaOr8 or TaORco coding sequences was performed with antennal or maxillary palp-derived cDNA templates and the following primers: TaORco forward: 5′CACCATGAATGTTCAACCAACCAAG3′; TaORco reverse: TTACTTCAGCTGCACCAGCAC; TaOr8 forward: 5′CACCATGAGACTCAGAAAGATGAACG3′; TaOr8 reverse: 5′CTATTTCGGTCCATACATTGTT3′. Amplicons were cloned into the pENTRTM vector using the GatewayR directional cloning system  and subcloned into the Xenopus laevis expression destination vector, pSP64t RFA.RNA was isolated from antennae or maxillary palps of adult female http://dx.doi.org/10.6084/m9.figshare.1092617).Plasmids were purified using the The ZR Plasmid Miniprep™-Classic  and sequenced by Macrogen Europe . DNA and amino-acid sequences for TaOr8 and TaORco have previously been publishedR)-octenol (described below): 17 compounds from Sigma-Aldrich , including 1-hepten-3-ol (CAS 4938-52-7), 3-methylbutanol (CAS 123-51-3), E-2-hexen-1-al (CAS 6728-26-3), heptaldehyde (CAS 111-71-7), octanal (CAS 124-13-0), propyl-acetate (CAS 109-60-4), 3-octanone (CAS 106-68-3), 6-methyl-5-hepten-2-one (CAS 110-93-0), 2,4,5-trimethylthiazole (CAS 13623-11-5), diallyl-sulfide (CAS 2179-57-9), benzaldehyde (CAS 100-52-7), indole (CAS 83-34-1), histamine (CAS 51-45-6), (+)-limonene oxide (CAS 203719-54-4), geranyl-acetate (CAS 105-87-3), (+)-fenchone (CAS 4695-62-9), 2-oxopentanoic acid (CAS 1821-02-9); 7 compounds from Merck , including methyloctanoate (CAS 111-11-5), ethyl-hexanoate (CAS 123-66-0), 2-heptanone (CAS 110-43-0), dimethyl-sulfide (CAS 2179-57-9), tryptamine (CAS 61-54-1), octanoic-acid (CAS 124-07-2) and D-glucuronolactone (CAS 32449-92-6); 2 compounds from Acros Organics , including methyl-salicylate (CAS 119-36-8) and octopamine (CAS 770-05-8); and 2 compounds from Alfa-Aesar , including L-lactic acid (CAS 79-33-4) and δ-Decalactone (CAS 705-86-2).For establishing the tuning curve, we used the following 29 chemicals, including  were obtained from Sigma. (R)-(−)-1-octen-3-ol  and (S)-(+)-1-octen-3-ol  chiral compounds were gifts from Bedoukian Research Inc.Racemic octenol (CAS number 3391-86-4) and ® SP6 Transcription Kit (ThermoFisher Scientific) and linearized pSP64tRFA expression vectors. Stage V-VI oocytes were manually separated and enzymatically defolliculated using a 2 mg/mL collagenase  solution  for 30 min at 18 °C. Oocytes were then successively washed in calcium-free ND96 and gentamycin-supplemented  calcium-free ND96. Oocytes were then washed and incubated in ND96 buffer supplemented with calcium (0.1 M), 5% heat-inactivated horse serum (ThermoFisher Scientific), 50 mg/ml tetracycline (Carl Roth GmbH), 100 mg/ml streptomycin  and 550 mg/ml sodium pyruvate  for four to five days. Oocytes were injected with 27.6 nL (27.6 ng of each cRNA) of RNA using the Nanoliter 2010 injector . Odorant-induced currents of oocytes expressing TaOr8 and TaORco were recorded using the two-microelectrode voltage-clamp technique (TEVC). The OC-725C oocyte clamp  maintained a −80 mV holding potential.The methodologies and protocols used in this study have been described elsewhereR), (S) or (RS)-octenol alone (10−10 M to 10−3 M). To measure the effect of DEET on TaOR8, we used (10−7 M) (R)-octenol or a combination of (10−7 M) (R)-octenol and DEET (10−3 M) in 1% DMSO for 8 s. Current was allowed to return to baseline between drug administrations. Data acquisition and analysis were carried out with the Digidata 1550 A digitizer and pCLAMP10 software .For the establishment of concentration-response curves, oocytes were exposed to (R)-octenol and known to elicit physiological or behavioral responses in mosquitoes . All chemicals used were administered at 400 nM, which corresponds to the EC50 of (R)-octenol. All the data analyses were performed using GraphPad Prism 5 .The tuning curve was generated using a panel 29 odorants including .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In addition, co-transfection of G6PD siRNA and miR-1 sponge partially reversed miR-1 sponge-induced reductions in cell viability and neoplasm growth. These results suggest that miR-1 suppresses the development and progression of HR-HPV 16/18-infected cervical cancer by targeting G6PD and may be a promising novel therapeutic candidate.Ectopic glucose-6-phosphate dehydrogenase (G6PD) expression may contribute to tumorigenesis in cervical cancer associated with high-risk human papillomavirus (HR-HPV 16 and 18) infections. Here, we demonstrate that microRNA-1 (miR-1) in association with AGO proteins targets G6PD in HR-HPV-infected human cervical cancer cells. miR-1 inhibited expression of a reporter construct containing a putative G6PD 3′-UTR seed region and suppressed endogenous G6PD expression. Down-regulation of miR-1 increased G6PD expression in cervical cancer cells. Regression analysis revealed that miR-1 levels correlate negatively with the clinicopathologic features in HR-HPV 16/18-infected cervical cancer patients. miR-1 overexpression inhibited proliferation and promoted apoptosis in cervical cancer cells and reduced xenograft tumor growth in nude mice. Conversely, sponge-mediated miR-1 knockdown markedly increased viability and reduced apoptosis in cervical cancer cells and supported neoplasm growth. Restoration of G6PD expression partially reversed the effects of miR-1 overexpression both  In 2008, cervical cancer was the third most common malignant cancer, accounting for 9% of new cases , and the fourth leading cause of cancer-related death, accounting for 8% of such deaths, among women worldwide . PersistGlucose-6-phosphate dehydrogenase (G6PD) is the rate limiting enzyme in the pentose phosphate pathway (PPP). Functionally, G6PD supplies ribose and NADPH that support biosynthesis and antioxidant defense, and is thus involved in the oxidative stress response . G6PD leMicroRNAs (miRNAs) are small noncoding RNAs that bind via base pair interactions with the 3′-untranslated region (UTR) of target mRNA to either degrade the mRNA or repress its translation . miRNAs In this study, we demonstrate that miR-1 might suppress the development and progression of HR-HPV 16/18-infected cervical cancer by targeting G6PD.The effectiveness and specificity of miRNA transfections were evaluated using Northern blots Figure . Co-immuRIP-Chip experiments were then performed in both Hela and Siha cervical cancer cells to validate computational predictions. Anti-AGO antibody was used to pull down endogenous AGO-containing miRNP complexes and associated mRNAs after miRNA transfection.MiRNPs were co-IPed with anti-AGO antibodies bound to protein G agarose beads. RNA associated with AGO protein complexes was then isolated for microarray profiling to identify transcriptome-wide miR-1/133a/206 targets in cervical cancer cells. After confirming successful miR-1/133a/206 transfection, Affymetrix GeneChip microarrays were used to profile mRNAs associated with miRNPs following miR-1/133a/206 transfection.RIP-Chip consistently indicated that G6PD mRNA was more strongly incorporated into miRNPs following miR-1 transfection than the other mRNAs examined Figure . By contTo further examine whether miR-1 directly targets G6PD mRNA in HR-HPV 16/18-infected (+) cervical cancer cells, G6PD expression was measured using qRT-PCR and Western blot in Hela and Siha cells transfected with miR-1 overexpression or control vectors. Databases were subsequently used to identify the potential target region of miR-1 in the G6PD mRNA 3′-UTR.P < 0.01) and by 65% in Siha  cells overexpressing miR-1. Treatment with plenti-G6PD partially restored G6PD expression in both Hela-plenti-miR-1 and Siha-plenti-miR-1 cells.G6PD mRNA expression was down-regulated by 71% in Hela   Figure . G6PD-si) Figure  and 3C. P < 0.01), but not with the G6PD 3′-UTR-mut plasmid (P > 0.05)  Figure . To veri) Figure . These dAll 60 patients with pathologically diagnosed cervical cancer were HPV DNA-positive (identified by PCR), and 88.33% (53/60) of these patients were positive for HR-HPV 16/18. The age range for these patients was 38 to 71 years, with a median age of 48 years. 18.1% had multiple HPV infections, and HPV16 infection was the most prevalent type (38.8%), followed by HPV-18 (35.1%), HPV-31 (9.2%), HPV-52 (6.3%), HPV-39 (5.5%), and HPV-58 (5.1%).Fifty-seven histopathologically-confirmed cervical cancer specimens were obtained from these 60 patients. The remaining three samples were necrotic and unsuitable for further analysis.P = 0.000000; 0.27 ± 0.05 in Siha vs. 1.59 ± 0.31 in C33A, P = 0.000001) and H8 cells . However, neither miR-133a nor miR-206 expression differed in HR-HPV+ cervical cancer cells compared to control cells , increased cell differentiation , and tumor diameter .Tumor formation was measured in mice after injections of different cervical cancer cells. Neoplasms formed last and grew slowest in mice treated with plenti-miR-1-transfected cells compared to the other treatment groups (plenti-miR-1 and plenti-miR-1 + G6PD control groups). Tumor growth was fastest in mice treated with miR-1 sponge-treated cervical cancer cells . However, partial G6PD knockdown counteracted miR-1 overexpression-induced growth .Tumors were larger in miR-1 sponge-treated groups than in the other groups 16 days post-injection  miR-1 bound to the 3′-UTR seed region of G6PD mRNA; ii) decreased miR-1 expression in HR-HPV 16/18-infected cervical carcinoma was correlated with carcinogenic development; iii) overexpression of miR-1 down-regulated G6PD, reduced proliferation, and promoted apoptosis in HR-HPV16/18+ cervical cancer cells; iv) co-transfection of both G6PD siRNA and miR-1 sponge partially reversed miR-1 sponge-induced promotions in cell viability and neoplasm growth. Taken together, these results indicate that miR-1 might suppress the development and progression of HR-HPV-16/18+ cervical cancer by directly targeting G6PD, and that miR-1 might therefore be a valuable novel therapeutic candidate.Animal use and care were in conducted in accordance with guidelines provided in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and in the Care and Use Guidelines of Experimental Animals established by the Research Ethics Committee of Kunming University of China (Permit Number: kmu-eac-2015117). All surgical procedures were performed under chloral hydrate anaesthesia, and all efforts were made to minimize suffering.This study also complied with the Ethical Declaration and was approved by the Human Ethics Committee and the Research Ethics Committee of Kunming University of China (Permit Number: kmu-hec-2014036). According to these guidelines, patients were informed that resected specimens would be kept by the Third People's Hospital of Yunnan Province and might be used for scientific research and that their privacy would be maintained. All of the patients who participated in this study provided informed consent.HPV-negative C33A (HPV-C33A), HPV18-positive Hela (HPV18+ Hela), and HPV16-positive Siha (HPV16+ Siha) human cervical carcinoma cells lines, as well as normal human cervical epithelial H8 cells, were used in this study. All cell lines were purchased from the Institute of Biochemistry and Cell Biology, Shanghai, China. Cells were maintained in Dulbecco's modified Eagle medium  supplemented with 10% fetal calf serum  at 37°C in a humidified incubator with 5% CO2. All cells were cultured at 70–80% confluence.To determine whether miR-1 contributed to carcinogenic events in HR-HPV-infected cervical cancer by targeting G6PD, miR-1 overexpression and/or miR-1 inhibition was established in cultured cervical cancer cells with or without G6PD overexpression. Proliferation and apoptosis were then examined in these cells using 3- -2, 5- diphenyltetrazolium bromide (MTT) and flow cytometry (FCM) assays, respectively.A lentiviral system was used for miR-1 overexpression. Hela and Siha cells were co-transfected with plenti-miR-1 along with the packaging plasmids psPAX2 and pMD2.G (Addgene), respectively (named Hela-plenti-miR-1 and Siha-plenti-miR-1). In control cells, plenti-miR-1 was replaced by control lemiR (named Hela-lemiR and Siha-lemiR). We simultaneously used miRNA sponge technology to inhibit miR-1 activity in cultured cervical cancer cells , 25. CulAdditionally, Hela/Siha-plenti-miR-1 was co-transfected with plenti-G6PD using the lentiviral overexpression system described above (named Hela/Siha-plenti-miR-1 + plenti-G6PD). Hela/Siha-plenti-miR-1 co-transfected with lemiR served as a control (named Hela/Siha-plenti-miR-1 + G6PD control). Similarly, Hela/Siha-miR-1-sponge was co-transfected with G6PD-siRNA plasmid; co-transfection with Empty-siRNA served as a control .To identify miRNAs that might target G6PD, the TargetScan, miRanda, and Diana microT computational algorithms were used . PotentiWe performed anti-AGO co-immunoprecipitation (co-IP) and downstream Affymetrix microarray analyses (“RIP-Chip”) to confirm that the computationally-predicted miRNAs targeted G6PD.6/plate. After 24 hours, cells were transfected with 25 nM “Pre-miRNA” (Ambion) for has-miR-1, has-miR-133a, has-miR-206, or Negative Control  using RNAiMAX  according to the manufacturer's instructions [For RIP-Chip transfections, Hela and Siha cells were plated in 10-cm culture plates at a density of 2.5 × 10The RIP-Chip co-IP assay has previously been described in detail , 27. BriMicroarray analysis of RNAs isolated via co-IP was performed using an Affymetrix Human Gene 1.0 ST chip at the University of Kentucky Microarray Core Facility. Eight biological replicates from three individual experiments were performed for each transfection condition.Northern blot analyses were performed using RNA isolated from Hela and Siha cells 48 h after transfection with miRNAs as described previously .After carefully rinsing in cooled PBS, cells were homogenized on ice in TRIzol . Total RNA was isolated using TRIzol according to the manufacturer's instructions. RNA quality and quantity were measured using a Nanodrop spectrophotometer , and RNA integrity was determined by gel electrophoresis.To detect miRNAs, 100 ng of RNA was labeled and hybridized using the Human microRNA Microarray Kit (Rel. 12.0)  according to the manufacturer's protocol. miR-1/133a/206 and control miRNAs were measured in eight biological replicates in three separate batches. Agilent miRNA microarrays (Version 1.0) were used. Hybridization signals were detected with an AgilentDNA G2505B microarray scanner, and scanned images were analyzed using Agilent feature extraction software (v10.10.1.1). All data were deposited in the NCBI Gene Expression Omnibus (GEO Series accession number GSE71953).Wild-type (wt) and mutant (mut) human G6PD mRNA 3′-UTR seed regions, which included the potential target site for miR-1, were cloned. The cloned sequences were inserted downstream of the pGL3 luciferase reporter gene to generate the G6PD 3′-UTR-wt and G6PD 3′-UTR-mut vectors. Briefly, 293T cells were seeded in 96-well plates and co-transfected with 100 ng/mL of the individual pGL3-G6PD 3′-UTR-wt/mut vectors and 50 nM miR-1 mimics or NC . Forty-eight hours after transfection, the effects of miR-1 treatment on luciferase activity were measured using a dual-luciferase reporter assay system kit (Promega) and a Tecan M200 luminescence reader  according to the manufacturer's instructions. Values were double-normalized to firefly luciferase activity and to cells transfected with empty control vectors .Sixty cervical cancer patients infected with HPV who were treated at the Third People's Hospital of Yunnan Province between February and December 2014 participated in the study. All patients underwent operations at our hospital. Samples from three of the patients were necrotic and were unsuitable for analysis. The remaining fifty-seven samples were collected and cervical tumors were histopathologically confirmed. Matched normal adjacent tissues at least 1 cm distal to the tumor margins were also collected. Specimens were immediately stored at −80°C until nucleic acids and proteins were isolated.Cervical cell samples were collected for HPV detection as described previously .-ΔΔCT) analysis method.miRNA was quantified using reverse transcription and quantitative real-time PCR (qRT-PCR). Small RNAs from cell and tissue samples were prepared using a TRIzol and miRNeasy mini kit  according to the manufacturer's instructions. cDNA was synthesized from total RNA using gene-specific primers according to the TaqMan MicroRNA Assay protocol provided by the manufacturer (Applied Biosystems). Quantitative PCR for miR-1/133a/206 was performed using an Applied Biosystems 7300 Sequence Detection system. The 10 μL PCR reaction contained 0.67 μL of reverse transcription product, 1 x TaqMan Universal PCR master mix, and 1 μL of primer and probe mix, according to the TaqMan MicroRNA Assay protocol (Applied Biosystems). Samples were normalized to snoRNA202 expression . Relativ-ΔΔCT method.Cells and tumor samples were also obtained for G6PD detection as described previously . The relG6PD protein expression was detected using Western blots in Hela and Siha cells transfected with miR-1 overexpression or sponge vectors according to a previously described protocol . BrieflyThe mouse anti-AGO antibody used for IP was also used for western blots at a dilution of 1:1000; the secondary rabbit anti-mouse HPR-conjugated secondary antibody was used at a dilution of 1:1500. Protein was visualized and quantities determined using an electrochemiluminescence (ECL) technique . Photographs were taken using the JS Gel Imaging System , and gray densities were calculated using SensiAnsys software .Cell viability was determined using the tetrazolium salt MTT assay as described previously . The optAn annexin V-FITC-flow cytometry assay kit (4A Biotech Co. Ltd.) was used to detect cellular apoptosis rates after G6PD-siRNA plasmid transfection as previously described .A total of 85 BALB/c strain nude mice  were housed and raised in the laboratory animal center of the Affiliated Cancer Hospital of Sun Yat-sen University. Animal use and treatment was approved by the Animal Ethics Committee of Sun Yat-sen University.6/mL) was injected intradermally into the left axilla of the mice. After seeding, liquid absorption at the injection site, tumor growth (volume and weight), and mouse survival were measured. Tumor volume was measured on days 5, 7, 12, 16, 19, 21, 25, and 27 post-injection. On day 27, all mice were sacrificed, tumors were isolated, and tumor weight and volume were determined. The largest (a) and smallest diameters (b) of each tumor were measured twice on days 5, 7, 12, 16, 19, 21, 25, and 27 to estimate tumor volume (V) using the formula V = 0.52 × a2 × b [Five mice were randomly assigned to each of the following 17 groups: normal human cervical epithelial H8 cells (H8)-treated group; miR-1 overexpression cervical cancer cell (Hela/Siha-plenti-miR-1)-treated groups; miR-1 deficient human cervical cancer cell (Hela/Siha-miR-1-sponge)-treated groups; matched control groups (Hela/Siha-lemiR and Hela/Siha-CX-control); G6PD rescue groups ; and G6PD inhibition groups . Cells in the log-phase growth stage were harvested and digested to obtain isolated cells using 0.25% pancreatin. Cells were washed with Dulbecco's Modified Eagle Medium (DMEM) without serum. A volume of 1 mL of each cell type , miRNA expression, and cervical cancer clinicopathologic characteristics, including clinical stage , cell grade , and tumor diameter (≤ 4 or > 4 cm). The strength of associations was determined using odds ratios (ORs) with 95% confidence intervals (CI); logistic regression was used to estimate adjusted ORs for ordinal data.