Ocimum basilicum Linn. var. pilosum (willd.)-Benth and Ocimum tenuiflorum var. CIM-AYU were subjected to GC–MS analysis and obtained 35 phytochemical constituents. Repellent potentiality of the Ocimum compounds was assessed against 3Q8I and 3N7H of Anophelesgambiae. PDB structures of mosquito odorant binding proteins were downloaded, processed and docking studies were performed along with reference ligand DEET using Schrodinger MAESTRO 9.2 software. Molecular docking results reveal that phenol, 2-methoxy-3-(2-propenyl)-, licopersin, gamma sitosterol and benzene, 1,2-dimethoxy-4-(2-propenyl)- from O. tenuiflorum var. CIM-AYU are strongly bound with 3N7H. Whereas, 4h-1-benzopyran-4-one, 5-hydroxy-6,7-dimethoxy-2-(4-methoxyphenyl)-, catechol and monoacetin from O. basilicum Linn. var. pilosum (willd.)-Benth. show high binding affinity with odorant binding protein 3Q8I. All natural compounds tested in the present study display better docking scores than DEET. The results further substantiate that the 12 out of 35 compounds of the two Ocimum species found to be ideal candidates for design and development of potential mosquito repellents. ADME properties of the tested compounds further confirm that bioactive compounds of Ocimum species were found to be in acceptable range. Synchronized application of at least two different natural compounds (with best docking scores) which target 3N7H and 3Q8I (Odorant Binding Proteins of mosquito) proteins may provide enhanced protection against mosquitoes bite. Based on the ADME properties, natural compounds of Ocimum species can be considered for design and development of safe mosquito repellents.Mosquitoes are exceptionally efficient in detecting their hosts for blood meal using odorant binding proteins, viz. 3N7H and 3Q8I and spread several dreadful diseases. DEET is a synthetic mosquito repellent widely used all over world for protection against mosquito bite. Reports reveal that, synthetic mosquito repellents may pose health problems in considerably large population. In view of the above fact, we made an attempt to discover efficient and novel natural mosquito repellent compounds with least impact on human health. Methanolic leaf extracts of The online version of this article (doi:10.1007/s13205-015-0346-x) contains supplementary material, which is available to authorized users. N,N-diethyl-3-methyl benzamide (DEET)  of A. gambiae. Further these studies will widen the scope to choose the most suitable compounds for design and development of effective and safe mosquito repellents.Studies confirm that plant extracts from Ocimum tenuiflorum var. CIM AYU and O. basilicum Linn. var. pilosum (willd)-Benth. Seeds were sown at K L University botanical garden. Fresh and healthy leaves were powdered, extracted in methanol, concentrated and finally the active components were analysed using GC–MS  and modified to make the protein biologically active and stable. Later Protein Preparation Wizard tool of Schrödinger’s software converts a raw PDB structure into all-atom fully prepared protein. In the modified protein, the active site was identified and further optimized by removing free water molecules and hetero atoms. For docking studies, only the ideal active site was selected. For favorable interactions between one or more ligand molecules on a receptor, receptor region with optimal binding affinity grid was generated for glide searches. Ligands obtained in all aspects follow Lipinski’s rule of 5. Structures obtained from Pubchem/Chemspider database were sketched using Chem sketch tool and finally the high quality all atom 3D structures were saved in Maestro (.mol) format. Optimization of structures were performed using LigPrep tool by removing unwanted molecules, addition of hydrogen, and ligand structure minimization. Finally, Grid-based Ligand Docking with Energetics (GLIDE) was performed.The 2D structures of 35 compounds of two Ocimum species were programmed in accordance with Lipinski’s rule of five. On the other hand, QikProp tool is used to evaluate the bioavailability of the lead molecules by assessing their physicochemical properties to observe the range of the Lipinski rule for induced molecules  and Rule of five and Rule of three  of potential mosquito repellent ligands were studied using QikProp module of the Schrödinger 9.2 software. ADME properties predicted for the potential compounds of O. tenuiflorum var. CIM AYU (20) and Ocimum basilicum Linn. var. pilosum (willd)-Benth. (15) yielded total of 35 compounds. Of the 35 compounds tested for their binding affinity with odorant binding proteins of Anopheles mosquito, 2-hexadecen-1-ol, phytol, dl-alpha-tocopherol, phenol-2-methoxy-3-(2-propenyl)-, lycopersin, gamma-sitosterol, benzene, 1,2-dimethoxy-4-(2-propenyl)- of O. tenuiflorum exhibit strong binding affinity with odorant binding protein 3N7H. Whereas, 4H-1-benzopyran-4-one, 5-hydroxy-6,7-dimethoxy-2-(4-methoxyphenyl), catechol, phytol, 2-hydroxy-6-methylbenzaldehyde, Monoacetin from O. basilicum Linn. var. pilosum (willd)-Benth display intense binding affinity with 3Q8I of A. gambiae -Benth. 5 yieldedOcimum species against mosquito odorant binding proteins (3Q8I and 3N7H) along with reference ligands are presented in Table O. basilicum Linn. var. pilosum (willd)-Benth., 2-hydroxy-6-methylbenzaldehyde exclusively bound to hydrophobic amino acid residue ALA 106 with backbone hydrogen bond with a glide score of −5.56. Moreover, this ligand exhibits additional pi–pi stacking interaction with PHE 123. All five compounds of O. basilicum show hydrogen bonding  and found to exhibit relatively higher G scores than reference ligand DEET  exhibit differential binding pattern with the compounds of O. tenuiflorum var. CIM AYU. Studies further reveal that the reference ligand DEET has affinity with 3N7H receptor protein and forms hydrogen bonding (−0.63) and selectively bound to ASN 56 amino acid residue with a glide score of −2.74. Of the seven compounds tested as against 3N7H, 2-hexadecen-1-ol, phytol and dl-alpha-tocopherol shown to have backbone hydrogen bonding by negatively charged GLU 49 amino acid residue with glide scores of −4.98, −4.64 and −4.54, respectively. Further the receptor pockets of these ligands were surrounded by hydrophilic and hydrophobic regions.Unlike 3Q8I, the odorant binding receptor protein of Ocimum species were found to be in the normal range - were found to interact with polar amino acid residue ASN 56 and recorded glide scores of −3.86 and −3.11, respectively. The ligand gamma-sitosterol establishes backbone hydrogen bonding and the receptor pocket is surrounded by hydrophobic region. Whereas benzene, 1,2-dimethoxy-4-(2 propenyl)- forms side chain hydrogen bonding and the pocket is enclosed by hydrophobic and hydrophilic areas correspondingly. Whereas phenol-2-methoxy-3-(2-propenyl)- and lycopersin also bound to ASN 56 amino acid residue but the former share with CYS 53 and the latter with LYS 29 amino acid residue. In contrast, ligand phenol-2-methoxy-3-(2-propenyl)- receptor pocket is totally surrounded by hydrophobic region. Interestingly, this ligand establishes backbone hydrogen bonding with hydrophobic (CYS 53) and Polar (ASN 56) amino acid residues respectively . Moreover, all compounds tested in the present study exhibit hydrogen bonding and also display hydrophobic activity with corresponding amino acids. Calculated ADME properties for all potential compounds of two Ocimum species, only 7 compounds -, licopersin, gamma sitosterol, and benzene, 1,2-dimethoxy-4- found potential against 3N7H with respect to glide score, H-bonding and amino acid residues. Whereas the other 5 compounds investigated against 3Q8I receptor, three compounds namely, 4h-1-benzopyran-4-one, 5-hydroxy-6,7-dimethoxy-2-(4-methoxyphenyl)-, catechol and monoacetin display higher G Score against 3Q8I when compared to phytol and 2-hydroxy-6-methylbenzaldehyde. The study further supports the earlier works of Ocimum plants used as mosquito repellents -Benth.] species. Of the 35 compounds tested from two Ocimum species, 12 compounds are properly docked with A. gambiae odorant binding proteins (3N7H and 3Q8I).In the present study, active sites of the receptor 3Q8I and 3N7H were determined and docked with the compounds obtained from GC–MS analysis from two MW (Molecular weight), QPlog Kp (Predicted skin permeability), donor HB (Estimated number of hydrogen bonds that would be donated by the solute to water molecules in an aqueous solution), accpt HB (Estimated number of hydrogen bonds that would be accepted by the solute from water molecules in an aqueous solution), according to Rule of Five Below is the link to the electronic supplementary material.


Drosophila melanogaster. Bcl-rambo mainly localized to the mitochondria of Drosophila Schneider 2 (S2) cells. The overexpression of Bcl-rambo, but not Bcl-rambo lacking a C-terminal transmembrane domain, induced apoptosis in S2 cells. Moreover, the ectopic expression of Bcl-rambo by a GAL4-UAS system induced aberrant morphological changes characterized by atrophied wing, split thorax, and rough eye phenotypes. Bcl-rambo induced the activation of effector caspases in eye imaginal discs. The rough eye phenotype induced by Bcl-rambo was partly rescued by the co-expression of p35, Diap1, and Diap2. By using this Drosophila model, we showed that human Bcl-rambo interacted genetically with Drosophila homologues of adenine nucleotide translocators and the autophagy-related 8 protein. The results of the present study demonstrated that human Bcl-rambo localized to mitochondria and at least regulated an apoptosis signaling pathway in Drosophila.Bcl-2 family proteins play a central role in regulating apoptosis. We previously reported that human Bcl-rambo, also termed BCL2L13, localized to mitochondria and induced apoptosis when overexpressed in human embryonic kidney 293T cells. However, the physiological function of Bcl-rambo currently remains unclear. In the present study, human Bcl-rambo was ectopically expressed in  Programmed cell death plays an essential role in the development and maintenance of tissue homeostasis in animals . ApoptosL), pro-apoptotic proteins , and BH3-only proteins  , an, an12], Drosophila melanogaster is commonly used as a model organism and is a powerful screening tool for investigating genetic interactions. Mammals and flies share some common features for apoptosis signaling pathways [Drosophila possesses apoptosis regulators, such as Bcl-2 family proteins, caspases, and inhibitor of apoptosis (IAP) proteins [Drosophila. The results obtained showed that Bcl-rambo localized to mitochondria. We also demonstrated that the ectopic expression of Bcl-rambo in transgenic flies induced apoptosis and caused aberrant morphological changes, including a rough eye phenotype.pathways –26. Drosproteins –29. In tDrosophila Schneider 2 (S2) cells were grown in Schneider's Drosophila medium  containing heat-inactivated fetal calf serum  and penicillin-streptomycin mixed solution  at 25°C. HEK 293T cells were maintained in DMEM medium (Life Technologies) containing heat-inactivated fetal calf serum and penicillin-streptomycin mixed solution at 37°C.Drosophila early embryos. Full-length Drob-1/Debcl and Buffy were inserted into pCR3-based expression vectors containing N-terminal FLAG or VSV tags. FLAG-tagged Drob-1 was inserted into pMT/V5-His A expression vectors.Human Bcl-rambo (1–485)  was descBcl-rambo and pUAST-Bcl-rambo (ΔTM) were injected into embryos to obtain stable transformant lines carrying UAS-Bcl-rambo and UAS-Bcl-rambo (ΔTM). P element-mediated germline transformation was accomplished as described [Bcl-rambo and UAS-Bcl-rambo (ΔTM), respectively.pUAST-escribed . F1 tranBcl-rambo and UAS-Bcl-rambo (ΔTM) fly lines were established in the present study. The fly lines carrying glass multiple reporter (GMR)-GAL4 were described previously [engrailed (en)-GAL4 driver fly line was kindly provided by Dr. Nicholas Dyson . UAS-GFP, UAS-p35, UAS-Diap1, GMR-Diap2, pannier (pnr)-GAL4, decapentaplegic (dpp)-GAL4, salivary gland (sg)-GAL4, Drob-1E26, Drob-1W105, Drob-1E26BuffyH37, Drob-1W105BuffyH37, BuffyH37, sesBorg, Ant2G0247sesBG0247, schlankG0061, and Atg8aEP362 fly lines were obtained from the Bloomington Drosophila stock center  and the Drosophila Genetic Resource Center . The GAL4 driver fly lines were crossed with the UAS fly lines, developed, and then reared at 28°C.Fly stocks were maintained at 25°C by standard food . The UAS-eviously . The engc , FLAG , HSP60 (insect) , α-tubulin , and VSV-G  were commercially obtained. Antibodies reactive to Cut (2B10), Discs large (4F3), Elav (7E8A10), and Prospero (MR1A) were obtained from the Developmental Studies Hybridoma Bank . Secondary antibodies conjugated to horseradish peroxidase (HRP) were purchased from Jackson ImmunoResearch .Antibodies reactive to Bcl-rambo , cytochrome ™ lipid reagent .S2 cells were transfected with expression vectors by HilyMax transfection reagent  or siLentfect® Red CMXRos  for 30 min prior to fixation. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethylketone  was used to prevent cell death. Tissues were dissected in phosphate-buffered saline (PBS). S2 cells and tissues were fixed with 4% paraformaldehyde (PFA)–PBS and treated with 0.1% and 0.3% Triton X-100–PBS, respectively. The permeabilized cells were blocked with normal goat serum and incubated with primary antibodies and secondary antibodies conjugated with Alexa Fluor 488 (Life Technologies). S2 cells and tissues were then stained with Hoechst 33342 and 4’,6-diamidino-2-phenylindole (DAPI) at final concentrations of 7.5 μM and 1.5 μg/ml, respectively. Samples were analyzed by the confocal laser scanning microscope FV10i .S2 cells were incubated with MitoTracker4 (500 μM) for 24 h. The cells were washed with PBS and disrupted by a dounce homogenizer (15 strokes) in Hepes-sucrose buffer containing 250 mM sucrose, 10 mM Hepes-KOH (pH 7.4), 1 mM EDTA, 1 mM EGTA, and the protease inhibitor cocktail Complete™ . Homogenates were centrifuged  to remove nuclei and unbroken cells. Supernatants were collected as post nuclear lysates (PNL) and further centrifuged  for separation into precipitates (P10 fraction) and supernatants. The supernatants were centrifuged  and separated into precipitates (P100 fraction) and supernatants (S100 fraction). Alternatively, S2 cells were washed with PBS and treated with digitonin lysis buffer (10 mM Hepes-KOH (pH 7.2), 100 μM digitonin, 250 mM sucrose, 50 mM NaCl, 5 mM EGTA, 2 mM MgCl2, 1 mM dithiothreitol, Complete™) on ice for 15 min. Cell lysates were centrifuged  to separate supernatants as cytosolic fractions. Precipitates were treated with Triton X-100 lysis buffer (50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 2 mM dithiothreitol, 2 mM sodium orthovanadate, and Complete™) on ice for 15 min and centrifuged  to remove insoluble materials. Supernatants were collected as organelle fractions containing mitochondria.S2 cells were transfected with pMT/V5-His A expression vectors encoding Bcl-rambo and Bcl-rambo (ΔTM), and incubated with CuSO™. They were homogenized with a dounce homogenizer in the presence of SDS sample buffer. After centrifugation , supernatants were collected. Protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes . The membranes were incubated with primary antibodies and then HRP-conjugated secondary antibodies. Protein bands were visualized using ECL Western Blotting Detection Reagents  and analyzed by ImageQuant LAS 4000 mini .Subcellular fractions were prepared as described above. Salivary glands were taken from third instar larva and heated at 95°C for 2 min in 0.11 mM Tris-HCl (pH 7.5) and CompleteS2 cells were stained with Hoechst 33342 (7.5 μM). Nuclear morphology was observed under a fluorescence light microscope . Apoptotic cells (%) were calculated as  x 100. In parallel with the apoptosis assay, transfection efficiency was evaluated by transfection with expression vectors encoding DsRed-monomer.Adult wings were dehydrated with isopropanol and mounted in Hoyer’s medium. Samples were observed under a light microscope SZX12 (Olympus) or SEM VE-7800  in the low vacuum mode. The phenotypes of the eye and thorax from at least four adult female flies (3 to 5 d old) were observed. The phenotypes of the wings from 4–5 adult female flies (3 to 5 d old) were observed. In these experiments, no significant variations were observed in eye, thorax, and wing phenotypes among the individuals. The L3-L4 area of the wings was quantified by Image J software. The mean of the L3-L4 area was calculated from 4–5 flies and normalized by the mean of the control L3-L4 area. The L3-L4 area (%) was calculated as the mean ± S.E. of three independent experiments. The brightness and contrast of all images taken by the light microscope were adjusted uniformly using Microsoft Office Picture Manager, which was applied equally to all images in each figure.™ Caspase-3/7 Green Detection Reagent (Life Technologies) at the final concentration of 5 μM for 1 h at 37°C. Eye imaginal discs were fixed with 4% PFA–PBS and permeabilized with 0.5% Triton X-100–PBS, followed by staining with Hoechst 33342 (5 μM). Samples were analyzed by confocal laser scanning microscopy.Third instar larvae were dissected in PBS, and imaginal discs were suspended in Grace’s insect medium in the presence of Cellevent™). Postnuclear lysates were precleared with Sepharose 6B for 1 h and then immunoprecipitated with anti-FLAG M2 affinity gels (Sigma-Aldrich) for 3 h. The immunoprecipitates were washed several times with Nonidet P-40 lysis buffer and analyzed by Western blotting.HEK293T cells were transfected with expression vectors by the calcium phosphate method and incubated for in the presence of Z-VAD-fmk (20 μM) for 16 h. Cells were solubilized in Nonidet P-40 lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, and 2 mM sodium vanadate, CompleteStatistical analyses were performed using an analysis of variance followed by the Tukey’s test for multiple comparisons.Drosophila S2 cells in order to examine whether the C-terminal TM of Bcl-rambo was necessary for localization to mitochondria in Drosophila. Confocal microscopy observations revealed that Bcl-rambo primarily co-localized with MitoTracker , but not Bcl-rambo lacking the C-terminal TM (1–459), localized to mitochondria . Bcl-ramoTracker . In contoTracker . ConsistoTracker . The locoTracker , indicatoTracker . These rBcl-rambo, pUAST-Bcl-rambo (ΔTM) or pUAST-Bcl-rambo (TM) together with pAct5C-GAL4 to investigate whether Bcl-rambo induced apoptosis in Drosophila. We found that Bcl-rambo significantly induced apoptosis, while Bcl-rambo (ΔTM) or Bcl-rambo (TM) only very weakly induced apoptosis  promoter in the dpp-GAL4 driver line [dpp-GAL4 driver fly lines induced morphological changes in a wing vein   at the protein level was almost equivalent in the sg-GAL4 driver fly lines  . The en observed . In contobserved . In Drosly lines . Thus, tGMR-GAL4 driver fly lines induced a rough eye phenotype, such as a reduction in eye size and the loss of ommatidia, bristles, and pigmentation  was expressed (data not shown). These results suggest that Bcl-rambo does not induce apoptosis in the pupal retinae at this period.In order to determine whether Bcl-rambo affected eye development, eye imaginal discs were stained with Elav for a marker of all photoreceptor cells, Prospero for a marker of R7 cells, and Cut for a maker of cone cell precursors. Elav, Prospero, and Cut were expressed in eye imaginal discs even when Bcl-rambo or Bcl-rambo (ΔTM) was expressed . These rDrosophila has two Bcl-2 family proteins: Drob-1/Debcl and Buffy/dBorg-2, both of which have been classified into pro-apoptotic multidomain proteins, and are closely related to mammalian Bok [Drob-1E26 or Drob-1W105 mutation or by the Drob-1E26BuffyH37 or Drob-1W105BuffyH37 mutation , Schlank (a Drosophila homologue of ceramide synthases), and Drosophila Atg8a. The loss-of-pigment phenotype induced by Bcl-rambo was effectively rescued by the sesBorg or Ant2G0247sesBG0247 mutation  flies did not exhibit the clear effects observed in Atg8aEP362 flies , mainly localized to mitochondria in 3T cells , and indno acids . This cono acids . Drosophnd Buffy –35. Drobll lines . Drob-1/lines [L . Thus, BDrosophila, Bcl-2 family members were found to not play a central role in the regulation of apoptosis [Drosophila S2 cells and caspase activation in eye imaginal discs. This is consistent with our previous findings in which Bcl-rambo induced caspase activation in HEK293T cells [c into the cytosol. These results suggest that Bcl-rambo regulates the mitochondrial signaling pathway of apoptosis.In poptosis ,26. Drobpoptosis . The ectpoptosis –35, indipoptosis . A previpoptosis . These f3T cells . In S2 cDrosophila eye and wing [reaper-induced mitochondrial defects [Drosophila eye [Drosophila. We showed that Bcl-rambo did not interact with anti-apoptotic or pro-apoptotic Bcl-2 family members in HEK293T cells [Drosophila. In addition, Bcl-rambo did not bind to Drob-1/Debcl or Buffy in HEK293T cells. Since the BHNo domain of Bcl-rambo was previously shown to induce caspase activation in HEK293T cells [Drosophila.Human Bcl-2 and murine Bax have been shown to play roles in the regulation of apoptosis in the and wing . Human B defects . Furtherhila eye . These f3T cells . Consist3T cells , the BHNLegionella protein SidF [Drosophila, adenine nucleotide translocators are encoded by sesB as a major isoform and the second isogene Ant2 [Drosophila member of the ceramide synthase family and controls growth and body fat [Drosophila.Bcl-rambo has been reported to regulate apoptosis by interacting with adenine nucleotide translocators , ceramidein SidF . Adenineein SidF . In Drosene Ant2 . Ceramidene Ant2 ,54. In sbody fat . Due to Drosophila Diap1 and Diap2 have been shown to inhibit caspases in order to prevent apoptosis [Drosophila, accompanied by a reduction in eye size and the loss of eye pigment. Although the reduced eye size caused by Bcl-rambo was only weakly rescued by p35, Diap1, and Diap2, reduced pigmentation was rescued by p35, Diap1 and Diap2. The shapes of cone cells and pigment cells were markedly altered by Bcl-rambo, and their morphologies were also partially rescued by p35 in the pupal retinae. Thus, the overexpression of Bcl-rambo may have multiple effects on cells, and the loss of cells may be secondary to defects rescued by p35. Previous studies reported that the ectopic expression of Drob-1/Debcl resulted in the rough eye phenotype [Drosophila S2 cells [Drosophila development [Drosophila.The baculovirus p35 and poptosis ,42. Bcl-henotype ,34, whilhenotype . Drob-1/S2 cells . These felopment ,57. Drobelopment . Thus, tDrosophila even though amino acid homology is small. A recent study showed that Bcl-rambo had a short WXXL/I motif and was a mammalian homologue of yeast Atg32 that mediated mitophagy and mitochondrial fragmentation [Drosophila. Thus, humans and Drosophila may have a similar pathway regulated by Bcl-rambo. By using Drosophila as a screening system, we revealed that human Bcl-rambo interacted genetically with Drosophila homologues of adenine nucleotide translocators and Atg8, suggesting that Bcl-rambo mediates at least two different pathways for apoptosis and mitophagy in Drosophila. These results indicate that the Drosophila model established in the present study can be a powerful screening tool for investigating genetic interactions. The further identification of gene(s) interacting with Bcl-rambo may help to address its physiological function in humans.In conclusion, the ectopic expression of Bcl-rambo induced the rough eye phenotype in transgenic flies. Bcl-rambo induced the rough eye phenotype, which suggested that a functional homologue of Bcl-rambo may exist in entation . Bcl-ramentation ,21,23, aS1 FigBcl-rambo (1–441), or pMT-V5-His A/Bcl-rambo (1–459) and then incubated in the presence of CuSO4 (500 μM) and Z-VAD-fmk (20 μM) for 24 h. S2 cells were stained for Bcl-rambo (green) and with DAPI (blue) and MitoTracker® Red (red). The stained cells in at least five different fields were observed by confocal laser scanning microscopy. Optical sections containing a single transfected cell are shown. Data were representative of two independent experiments. Scale bars indicate 10 μm.(A) Structures of human Bcl-rambo and its mutants. (B) S2 cells were transfected with pMT-V5-His A, pMT-V5-His A/(TIF)Click here for additional data file.S2 FigGAL4 together with pMT-V5-His A, pMT-V5-His A/Bcl-rambo, pMT-V5-His A/Bcl-rambo (ΔTM), pMT-V5-His A/Bcl-rambo (1–441), pMT-V5-His A/Bcl-rambo (1–459), or pUAST-DsRed-monomer in the presence of CuSO4 (500 μM) for 24 h. Cells were stained with Hoechst 33342. Nuclear morphology was observed by fluorescent microscopy. Apoptotic cells (%) are shown as the mean ± S.E. of three independent experiments. **P<0.01, significantly different from the control. n.s., not significant. Transfection efficiency was measured by counting DsRed monomer-expressing cells, and calculated to be 16.5 ± 0.4% (the mean ± S.E of three independent experiments).(A) S2 cells were transfected with pAct5C-(TIF)Click here for additional data file.S3 FigGMR-GAL4 driver fly lines. (A)  GMR-GAL4/w; +; UAS-Bcl-rambo/+ (strain #1),  GMR-GAL4/UAS-Bcl-rambo; +; + (strain #6),  GMR-GAL4/w; UAS-Bcl-rambo/+; + (strain #46) (B)  GMR-GAL4/w; UAS-Bcl-rambo (ΔTM)/+; + (strain #10),  GMR-GAL4/w; UAS-Bcl-rambo (ΔTM)/+; + (strain #26),  GMR-GAL4/UAS-Bcl-rambo (ΔTM); +; + (strain #148) The morphology of adult eyes was observed by light microscopy (a–c) and SEM (d–i). Scale bars in d–f and g–i indicate 50 μm and 14.2 μm, respectively. Data were representative of two independent experiments.(A and B) Bcl-rambo and Bcl-rambo (ΔTM) were expressed using (TIF)Click here for additional data file.S4 FigGMR-GAL4 driver fly lines. (A)  GMR-GAL4/+; UAS-Bcl-rambo/UAS-GFP; +,  GMR-GAL4/w; UAS-Bcl-rambo/+; UAS-p35/+,  GMR-GAL4/w; UAS-Bcl-rambo/+; UAS-Diap1/+,  GMR-GAL4/w; UAS-Bcl-rambo/+; GMR-Diap2/+. (B)  GMR-GAL4/+; UAS-Bcl-rambo (ΔTM)/UAS-GFP; +,  GMR-GAL4/w; UAS-Bcl-rambo (ΔTM)/+; UAS-p35/+,  GMR-GAL4/w; UAS-Bcl-rambo (ΔTM)/+; UAS-Diap1/+,  GMR-GAL4/w; UAS-Bcl-rambo (ΔTM)/+; GMR-Diap2/+. The morphology of adult eyes was observed by light microscopy (a–d) and SEM (e–l). Scale bars in e–h and i–l indicate 50 μm and 14.2 μm, respectively. Data were representative of two independent experiments.(A and B) Bcl-rambo and Bcl-rambo (ΔTM) were expressed using (TIF)Click here for additional data file.S5 FigGMR-GAL4 driver fly lines. (A)  GMR-GAL4/w; UAS-Bcl-rambo/+; +,  GMR-GAL4/w; UAS-Bcl-rambo/BuffyH37; +,  GMR-GAL4/w; UAS-Bcl-rambo (ΔTM)/+; +,  GMR-GAL4/w; UAS-Bcl-rambo (ΔTM)/ BuffyH37; +. The morphology of adult eyes was observed by light microscopy (a–d) and SEM (e–l). Scale bars in e–h and i–l indicate 50 μm and 14.2 μm, respectively. Data were representative of two independent experiments.Bcl-rambo and Bcl-rambo (ΔTM) were expressed using (TIF)Click here for additional data file.


T1 mapping and ECV imaging are promising diagnostic tools in patients with suspected myocarditis. However, there are currently no data on the potential influence of slice orientation on the derived T1 and ECV values. Thus, we compared global myocardial T1 and ECV values between slices with short and long axis orientation.This study included 50 patients with clinically suspected myocarditis, who underwent CMR at 1.5 Tesla. T1 quantification was performed on three short  and on three long axis slices , respectively. The modified Look-Locker inversion-recovery (MOLLI) sequence was used before and 15 minutes after administration of 0.075 mmol/kg Gadolinium-BOPTA. Native T1, post-contrast T1 and extracellular volume (ECV) maps were calculated using a dedicated plug-in written for the OsiriX software. Mean native and post-contrast T1 as well as ECV were obtained from the three short and long axes, respectively.There were significantly lower median native myocardial T1 values on long axis slices (1079 (1040-1130.0)) compared to short axis slices (1103 (1069-1140); p=0.0026). However, there were no significant differences in post-contrast myocardial T1 values (median 554 (505-596) vs. 555 (509-601); p=0.2299) or ECV (median 30 (27-33) vs. 29 (26-33); p=0.6063) between short and long axis slices, respectively.Slice orientation could be a potential confounding factor for the assessment of native global myocardial T1 values in myocarditis. Possible explanations for this finding include technical issues, such as variations in through-plane motion or partial-volume effects related to slice orientation, but also the heterogeneity of myocardial injury in myocarditis. However, our data indicate that global myocardial ECV values could be less sensitive to slice orientation.


Neurospora crassa, a five-protein complex, DCDC, catalyzes H3K9 methylation, which then directs DNA methylation. Here, we identify and characterize a gene important for DCDC function, dim-3 (defective in methylation-3), which encodes the nuclear import chaperone NUP-6 (Importin α). The critical mutation in dim-3 results in a substitution in an ARM repeat of NUP-6 and causes a substantial loss of H3K9me3 and DNA methylation. Surprisingly, nuclear transport of all known proteins involved in histone and DNA methylation, as well as a canonical transport substrate, appear normal in dim-3 strains. Interactions between DCDC members also appear normal, but the nup-6dim-3 allele causes the DCDC members DIM-5 and DIM-7 to mislocalize from heterochromatin and NUP-6dim-3 itself is mislocalized from the nuclear envelope, at least in conidia. GCN-5, a member of the SAGA histone acetyltransferase complex, also shows altered localization in dim-3, raising the possibility that NUP-6 is necessary to localize multiple chromatin complexes following nucleocytoplasmic transport.Heterochromatin and associated gene silencing processes play roles in development, genome defense, and chromosome function. In many species, constitutive heterochromatin is decorated with histone H3 tri-methylated at lysine 9 (H3K9me3) and cytosine methylation. In  Neurospora crassa is decorated with DNA methylation and histone H3 lysine 9 (H3K9) methylation, but unlike the case in mammals, loss of these epigenetic marks does not compromise viability. In Neurospora, the DCDC, a five-member Cul4-based protein complex, trimethylates H3K9. Little information is available on the regulation of DCDC or similar complexes in other organisms. Using forward genetics, we identified a novel role for Importin α (NUP-6) for the function of DCDC. Although NUP-6 typically functions in nucleocytoplasmic transport, the dim-3 strain, which contains an altered nup-6 gene that reduces DNA methylation and H3K9me3, shows normal nuclear transport of the heterochromatin machinery and a canonical transport substrate. Two DCDC members are mislocalized from heterochromatin in the dim-3 mutant, signifying that NUP-6 may be important for targeting key proteins to incipient heterochromatic DNA. The euchromatic complex SAGA has increased euchromatin localization in dim-3, suggesting that NUP-6 may localize multiple chromatin complexes to sub-nuclear genomic targets.The epigenetic information contained in chromatin is essential for development of higher organisms, and if misregulated, can lead to the unregulated growth associated with human cancers. Chromatin is typically classified into two basic types: gene-rich 'euchromatin', and gene-poor heterochromatin, which is also rich in repeated DNA and 'repressive chromatin marks'. As in humans and eukaryotes generally, heterochromatin in  Neurospora crassa does not require DNA methylation for viability  Total (T) extract, as well as the cytoplasmic (C) and nuclear (N) fractions from strains expressing NUP-6+-3xFLAG or NUP-6dim-3–3xFLAG, as well as a strain lacking any 3xFLAG-tagged protein, were analyzed by standard western procedures, blotting for the FLAG tagged protein as well as control cytoplasmic  and nuclear (histone H3) proteins. Quantification of three separate experiments, with error shown, presented as the percent of cytoplasmic and nuclear NUP-6–3xFLAG are graphed below. [C] Isolated nuclei from strains expressing NUP-6+-3xFLAG and NUP-6dim-3–3xFLAG, as well as a strain lacking any 3xFLAG-tagged protein, were analyzed by standard western procedures, blotting for the FLAG tagged protein or the histone H3 loading control. The reproducible higher mobility of NUP-6dim-3–3xFLAG might be indicative of post-translational modification(s), as Xenopus Importin α has been reported to have multiple phosphorylation sites [+-3xFLAG are shown below. [D] Linear growth rate  of four wild type Neurospora strains  and four dim-3 strains , as measured by race tube growth.[A] Structural model of NUP-6 Click here for additional data file.S3 Figam) region Southern blot assays (as in dim-3+ (WT) strain, along with methylation mutant  and WT controls. “dim-3” strain has both mutations reintroduced while “WT” control shows the effect of just the hygromycin targeting cassette.Heterochromatic (8:G3 or 2:B3) or euchromatic (ys as in  of indic(EPS)Click here for additional data file.S4 FigmC-sensitive enzyme AvaII to assess DNA methylation levels in a dim-3+ (WT) strain supplemented with histidine, or dim-3 strains supplemented with additional amino acids, as listed, or those same amino acids in tandem with histidine. Supplemented methionine presumably restores DNA methylation by increasing internal S-adenosylmethionine (SAM) levels, as shown previously  DIM-2-DAM or [B] HP1-DAM grown with or without histidine and probed for the heterochromatic region 8:A6 or the euchromatic region pan-1. Genomic DNA was digested with DpnI (DI), specifically cutting GAmTC sequences or with the Am- insensitive isoschizomer DpnII (DII) to monitor complete digestion.Southern blot assay of DamID experiments with (EPS)Click here for additional data file.S8 Figdim-3+   DamID experiments with dim-3+ (WT) and dim-3 expressing DIM-5-DAM grown in minimum medium (min) or medium containing histidine (+HIS). Δdim-7; DIM-5-DAM sample indicates a complete loss of DIM-5-DAM targeting [pan-1) are shown. [C] DamID experiments with DIM-7-DAM and DIM-9-DAM.[A] DamID Southern blot experiments of the heterochromatic region 8:G3, as in (EPS)Click here for additional data file.S12 Figccg::DIM-7-GFP and HP1-mCherry in a WT strain. One conidium with two nuclei is displayed. Scale bar indicates 5μm. (B) Representative differential interference contrast (DIC), GFP fluorescent, mCherry fluorescent, and merged images of Pccg::NCA-1-GFP (CAB65295.1), which marks the nuclear and cell membranes  α-GFP western blot showing the total culture, cytoplasmic fraction, and nuclear fraction levels of overexpressed DIM-7-GFP found in nuclei isolated from dim-3+ (WT) or dim-3 strains expressing this construct. PonceauS-stained membrane of proteins present at the dye front serves as a loading control.[A] α-GFP western blots of whole cell extract of either a (EPS)Click here for additional data file.S14 Figdim-3+ cells expressing NUP-6-GFP from the native promoter, or [B] overexpressed NUP-6-GFP using the ccg promoter in dim-3 cells. Each panel displays one conidium, with one to three nuclei. Densely staining DNA foci are thought to represent centromeric heterochromatin [–33 (X2-test).[A-B] Additional examples, relative to hromatin . Scale b(EPS)Click here for additional data file.S15 FigDifferential interference contrast (DIC), GFP fluorescent, mCherry fluorescent, and merged images of NUP-6-GFP and HP1-mCherry in a WT strain background. Yellow arrows mark potential co-localization sites. Four independent examples are shown, and scale bar indicates 5μm. For each panel, one conidium showing one or two nuclei is displayed.(EPS)Click here for additional data file.S16 Figdim-3 cells expressing NUP-6dim-3-GFP from the native promoter, as in dim-3-GFP using the ccg promoter in dim-3+ cells. Each panel displays one conidium, with one to three nuclei. Densely staining DNA foci are thought to represent centromeric heterochromatin  Additional examples of differential interference contrast (DIC), GFP fluorescent, Hoechst33342 stained DNA, and merged images of [A] hromatin . Scale b(EPS)Click here for additional data file.S1 MovieZ-stack image movie of NUP-6-GFP constructed with fluorescent images and compiled using ImageJ. One conidium with three nuclei is displayed.(AVI)Click here for additional data file.S2 Moviedim-3-GFP constructed with fluorescent images and compiled using ImageJ. One conidium with two nuclei is displayed.Z-stack image movie of NUP-6(AVI)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Myocarditis subsumes a variety of entities, including diverse courses from complete healing to dilated cardiomyopathy with severe myocardial fibrosis. T1-mapping cardiovascular magnetic resonance (CMR) has the ability to quantify myocardial extracellular volume (ECV) as a surrogate of acute and chronic myocardial injury. Galectin-3 is an important mediator of fibrogenesis and contributes to adverse left ventricular (LV) remodeling. This study evaluated, if myocardial ECV expansion is linked to Galectin-3 levels in patients with myocarditis.Galectin-3 blood levels were measured in 20 patients with myocarditis using a commercially available chemiluminescent microparticle immunoassay . T1 quantification was performed at 1.5 Tesla using the modified Look-Locker inversion-recovery (MOLLI) sequence before and 15 minutes after administration of 0.075 mmol/kg gadolinium-BOPTA. Global myocardial ECV was calculated from T1 maps generated by a dedicated plug-in written for the OsiriX software.Median Galectin-3 level was 17.4 ng/mL (interquartile range 13.2 to 20.5 ng/mL) and median global myocardial ECV was 29 % (interquartile range 26 to 33 %) in the study population. There was a significant correlation between Galectin-3 levels and global myocardial ECV . In contrast, no significant correlation was found between Galectin-3 levels and LV end-diastolic volumes , LV end-systolic volumes , LV stroke volumes ; LV ejection fractions , Troponin T levels  or NT-proBNP levels , respectively.Myokardial ECV expansion, as a surrogate for myocardial injury, is associated with increased Galectin-3 levels, indicating activated fibrogenesis in patients with myocarditis. Combining Galectin-3 measurements with ECV-imaging could improve risk stratification beyond conventional imaging parameters or biomarkers in these patients.Marija-Orlovic-Foundation.


Doxazosin inhibited the phosphorylation of VEGF receptor-2 (VEGFR-2) and downstream signaling, including PI3K, Akt, 3-phosphoinositide-dependent protein kinase 1 (PDK1), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor 1 (HIF-1α). However, it had no effect on VEGF-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, doxazosin reduced tumor growth and suppressed tumor vascularization in a xenograft human ovarian cancer model. These results provide evidence that doxazosin functions in the endothelial cell system to modulate angiogenesis by inhibiting Akt and mTOR phosphorylation and interacting with VEGFR-2.Doxazosin is an α1 adrenergic receptor blocker that also exerts antitumor effects. However, the underlying mechanisms by which it modulates PI3K/Akt intracellular signaling are poorly understood. In this study, we reveal that doxazosin functions as a novel antiangiogenic agent by inhibiting vascular endothelial growth factor (VEGF)-induced cell migration and proliferation. It also inhibited VEGF-induced capillary-like structure tube formation  Angiogenesis is an important but complex process that occurs during endothelial cell development, growth, and movement, as well as wound healing and endothelial cell-mediated degradation of the extracellular matrix. Multistep angiogenesis is vital for cell division and metastasis in most solid tumors. Angiogenesis, the constitution of new blood vessels forming from pre-existing vessels, occurs during embryonic development as well as invasive tumor growth and tumor pathogenesis , 2. AngiDoxazosin , a quinazoline compound, functions as an α1 adrenergic receptor blocker . It is uin vitro human umbilical vein endothelial cell (HUVEC) and in vivo mouse model systems. We also identified the molecular pathways responsible for Akt- and mammalian target of rapamycin (mTOR)-dependent endothelial cell growth during tumorigenesis. Doxazosin inhibited the phosphorylation of signaling modulators downstream of phosphatidylinositol-3'-kinase (PI3K) including Akt, phosphoinositide-dependent protein kinase 1 (PDK1), and mTOR by interacting directly with VEGFR-2. Therefore, this interaction results in potent antiangiogenic and antitumor effects. The current findings suggest that doxazosin plays a vital role in regulating cellular angiogenesis.Most studies on angiogenesis have focused on endothelial cell proliferation, migration, and capillary-like tubule formation. In the current study, we investigated the antiangiogenic effects of doxazosin in tumors using The migration of endothelial cells is essential during angiogenesis. Therefore, we explored whether doxazosin modulated the effects of VEGF on cell migration using a modified Boyden Transwell chamber system in HUVECs. As expected, VEGF promoted the migration of doxazosin-treated cells compared to control cells. Doxazosin inhibited migration in a dose-dependent manner, and the maximal effect was seen at a concentration of 20 μM Fig. . No migrin vitro antiangiogenic activity of doxazosin was analyzed by assessing its effects on the VEGF-stimulated proliferation of endothelial cells using [3H] thymidine incorporation. Doxazosin inhibited VEGF-induced HUVEC DNA synthesis significantly  ovarian carcinoma cells. Lysates from SKOV-3 cells treated with various concentrations (0-25 μM) of doxazosin were analyzed by Western blotting, which revealed that VEGF induced the phosphorylation of PI3K and Akt. As shown in Fig. In addition to Akt, doxazosin also decreased the phosphorylation of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) (Tyr37/46) Fig.  and PDK1the Akt/PDK1/mTOR complex. As shown in Fig. We next assessed the effects of doxazosin on downstream mediators of the PI3K/Akt pathways. Doxazosin dramatically diminished the phosphorylation of mTOR at Ser-2448 and p70 ribosomal S6 kinase (p70S6K) at Thr-421 Fig. . TherefoPI3K/Akt signaling not only mediates VEGF-induced cell proliferation and migration, but also the expression of VEGF and HIF-1α . ConsistVEGFR-2 transcription was then evaluated using a luciferase reporter assay system and a construct containing the VEGFR-2 promoter fused to luciferase. Luciferase activity decreased in response to treatment with doxazosin in a dose-dependent manner  to assess the biological and functional relevance of the direct relationship between VEGFR-2 and doxazosin. VEGFR-2 is a key signal transducer during VEGF-induced endothelial cell vascular development and pathological angiogenesis. As shown in Fig. ner Fig. , suggestin vivo. SKOV-3 ovarian cancer cells were injected subcutaneously into nude mice (10/group), and the tumors were allowed to grow for 12 days until they reached a mean volume of 100 mm3. The mice were treated orally with control or doxazosin, and tumor growth and morphology was evaluated every 3 days for 24 days. Doxazosin-treated tumors weighed ~75% less than those from control mice  staining revealed that tumors in the control group were high-grade carcinoma with an irregular cell distribution. In contrast, doxazosin-treated tumors exhibited large areas of late-apoptotic or necrotic cells Fig. . MoreoveIn this study, we identified a novel biological function of doxazosin as a powerful antiangiogenic modulator that functions via the Akt/mTOR signaling pathway. Quinazoline-derived α1-adrenoceptor antagonists exert anticancer activity in prostate cancer. In addition, the induction of apoptotic cell death and inhibition of angiogenesis by doxazosin have been reported widely -16. NevePI3K/Akt signaling plays a vital role in the biological functions of human malignant tumors. Recent studies have reported that suppressing PI3K might be beneficial to inhibit tumor angiogenesis , 22-24. In summary, our data demonstrate that doxazosin could suppress endothelial cell functions, including cell proliferation, migration, invasion, and capillary-like tubule formation, by suppressing VEGFR-2 phosphorylation and inhibiting Akt/mTOR signaling. It also suppresses the expression of HIF-1α and VEGF in ovarian carcinoma cells. These data supply additional evidence to support a role for doxazosin as a potent modulator of the biological and physiological mechanisms relevant to angiogenesis. The potential of doxazosin as an ovarian cancer treatment should be assessed in future studies.2 incubator. Primary HUVECs  were grown on 0.3% gelatin-coated dishes  in EGM-2 BulletKit medium (Clonetics). Rapamycin was purchased from Cell Signaling Technology . Doxazosin and all other chemicals were purchased from Sigma-Aldrich. The following primary antibodies were used: anti-phospho-VEGFR-2 (Y1175), anti-VEGFR-2, anti-phospho-PI3K, anti-PI3K, anti-phospho-Akt, anti-Akt, anti-HIF-1α, anti-phospho-ERK1/2, anti-ERK1/2 , anti-phospho-PDK1, anti-PDK1, anti-phospho-4E-BP1, anti-4E-BP1, anti-phospho-mTOR, anti-mTOR, anti-phospho-p70S6K, anti-p70S6K, PCNA, cyclin D1, survivin , anti-CD31 , anti-VEGF165 , and anti-β-actin (Sigma-Aldrich).Human ovarian cancer cell lines (SKOV-3 and OVCAR-3) were purchased from the American Type Culture Collection . They were grown in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin at 37°C in a humidified 5% CO3H]-Thymidine incorporation was assessed as described previously -Thymidine  was added 4 h prior to analysis. High-molecular-mass compounds with [3H]-radioactivity were then precipitated using 10% trichloroacetic acid for 1 h at 4°C. Cells were solubilized in 0.2 N NaOH containing 0.1% sodium dodecyl sulfate (SDS), and [3H] thymidine incorporation was calculated using a liquid scintillation counter . ATP for 15 min at 37°C. The reaction products were developed using autoradiography, and the radioactive lipids were quantified using liquid scintillation counting.eviously -30. BrieCells were rinsed with phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay (RIPA) buffer supplied with protease inhibitors. The concentration of protein samples was then evaluated using a Bradford protein assay kit . Equal amounts of protein were loaded onto 8-12% SDS-polyacrylamide gel electrophoresis (PAGE) gels and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). After blocking, the membranes were incubated at room temperature with primary antibodies for 1 h. They were then rinsed three times in wash buffer, followed by incubation with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. The protein bands were developed using an enhanced chemiluminescence (ECL) detection system.Tumor samples were collected from mouse xenografts and fixed in 10% neutral-buffered formalin (Sigma-Aldrich). Slides were then stained using H&E (Sigma-Aldrich) according to the manufacturer's instructions. For immunoblotting, protein lysates were prepared by homogenizing the frozen tumor tissues. Protein quantitation and immunoblotting were then performed as described above with 30-μg protein/sample. For immunohistochemistry, paraffin-embedded ovarian tumor tissues were serially sectioned into 5-μm slices. The prepared slides were then deparaffinized, rehydrated in xylene and graded alcohols, and then rinsed in PBS. The slides were also incubated in 5% hydrogen peroxide in methanol for 20 min to block endogenous peroxidase activity. Sections were incubated with a saturating concentration of anti-mouse CD31  (Abcam) antibody overnight at 4°C, followed by a streptavidin-peroxidase complex at room temperature for 1 h. Microvessel density (MVD) was quantified in five randomly selected individual tumor fields (at 40× magnification) per sample, and the number of microvessels was counted under a high-powered microscope (400× magnification). All immunochemical analyses were performed using an Axiophot 2 apparatus .t-test for comparisons between two groups. Data are presented as the means ± SDs, or SEM for triplicate experiments. Statistical significance was defined as P<0.05. Values with 95% confidence (P<0.05) are depicted with an asterisk (*) on each graph.Results were analyzed statistically using Student's


Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in  C. elegans to identify proteins that are important for early steps in the generation of dense-core vesicles, such as packaging the correct molecular cargos in the vesicles. We identify several factors that are conserved between worms and humans and point to a new role for a protein complex that had previously been shown to be important for controlling trafficking in other cellular compartments. The identification of this complex suggests new cellular trafficking events that may be important for the generation of dense-core vesicles.Animal cells package and store many important signaling molecules in specialized compartments called dense-core vesicles. Molecules stored in dense-core vesicles include peptide hormones like insulin and small molecule neurotransmitters like dopamine. Defects in the release of these compounds can lead to a wide range of metabolic and mental disorders in humans, including diabetes, depression, and drug addiction. However, it is not well understood how dense-core vesicles are formed in cells and package the appropriate molecules. Here we use a genetic screen in the microscopic worm  The dense-core vesicle is a specialized secretory organelle found in neurons and endocrine cells. In endocrine cells, these vesicles are often called secretory granules; for simplicity, we will here use the term ‘dense-core vesicles’ to refer to both the neuronal and endocrine carriers. Dense-core vesicles package several classes of cargo, including peptide hormones, growth factors, and biogenic amines. In response to elevated calcium, dense-core vesicles release their cargos to modulate a variety of biological processes ranging from blood glucose homeostasis to development, function and plasticity of the nervous system. Despite the importance of the signaling molecules released by dense-core vesicles, surprisingly little is known about how dense-core vesicles are generated and acquire their proper cargos –4.2+ milieu of the trans-Golgi . Lysates were passed 10 times through a 20G needle and incubated for 30 min at 4°C. Lysates were pre-cleared by a 20,000 g centrifugation at 4°C for 15 minutes and the supernatant was incubated with 30 μg of anti-GFP nanobody bound to magnetic beads, for two hours at 4°C. The beads were washed three times with 250 μL wash buffer . Samples were eluted in two steps with (1) 0.2 M glycine pH 2.5 and (2) 1 M NH4OH. Eluents were neutralized with 0.33 M Tris-HCl pH 7.5 and stored at -80°C.For immunoprecipitation, 4x10Samples were processed using modified FASP (filter aided sample preparation) to remove mass spec incompatible substances . SamplesA total of 0.15 μg of peptides was chromatographically separated onto a Thermo LTQ-Velos Pro mass spectrometer coupled with a Waters nanoACQUITY liquid chromatography system. A 75 μm fused silica column (Polymicro Technologies) was loaded with 30 cm C12 Jupiter (Phenomenex), 4 μm reverse-phase beads, and a 100 μm fused silica Kasil frit trap (PQ Corporation) loaded with 4 cm of C12 Jupiter. Peptides were separated by a 110 minute gradient consisting of buffer A  and buffer B . Two analytical replicates were run for each sample with each set of replicates randomized. Quality control samples were run every sixth sample and at the beginning of the runs to assess column chromatography stability.Rattus norvegicus FASTA database containing contaminants. False discovery rates were determined via a decoy database using Percolator [Data were searched with SEQUEST  against rcolator  at a q-vrcolator . Lists oFor co-immunoprecipitation, 832/13 cells were cultured and lysed as described above (Mass spectrometry). The lysates were incubated with 20 μg anti-GFP nanobody bound to magnetic beads, the beads were washed three times with lysis buffer and resuspended in Laemmli loading buffer. Samples were resolved on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose or PVDF membranes. To detect co-precipitated proteins, we added the following primary antibodies: mouse monoclonal anti-GFP , monoclonal anti-mCherry , monoclonal anti-VPS50 , rabbit polyclonal anti-SNAP29 , or monoclonal anti-beta-tubulin as a loading control . The secondary antibody was an Alexa Fluor 680-conjugated goat anti-mouse antibody  or Alexa Fluor 680-conjugated goat anti-rabbit antibody . A LI-COR processor was used to develop images.Worms were mounted on 2% agarose pads and anesthetized with 50 mM sodium azide. Images were obtained using a Nikon 80i wide-field compound microscope except for the images shown in 5 832/13 cells per well were seeded onto sterilized cover slips placed in 12 well cell culture plates. The next day, cells were rinsed twice with ice-cold PBS and fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature. The cells were rinsed twice with PBS and permeabilized with 0.5% Triton X-100 in PBS for 5 minutes at room temperature. The cells were again washed twice with PBS and then placed in 5% milk in PBS for 1 hour at room temperature, followed by immunofluorescence staining with rabbit polyclonal anti-CCDC132  and mouse monoclonal anti-GFP  in 0.5% milk in PBS at room temperature for 1 hour. The cells were then washed with PBS three times for 5 minutes each, and incubated with Rhodamine anti-rabbit secondary antibody  and Alexa Fluor 488 anti-mouse secondary antibody  at room temperature for 1 hour. The cells were washed with PBS three times for 5 min each and examined by fluorescence microscopy (Nikon 80i wide-field compound microscope). The anti-CCDC132 antibody recognizes VPS50.2x10P values were determined using GraphPad Prism 5.0d (GraphPad Software). Data sets with multiple comparisons were analyzed by a one-way ANOVA followed by a Bonferroni posthoc test to examine selected comparisons or by Dunnett’s test if all comparisons were to the wild type control. Pairwise data comparisons were analyzed by two-tailed unpaired t tests.S1 Figegl-30(tg26) has hyperactive locomotion and deep body bends. The egl-30(tg26) locomotion phenotype is suppressed by the eipr-1(ox316), vps-50(ok2627), vps-52(ox345), and vps-53(ox339) mutations. (B) eipr-1 acts in neurons to control egg laying. The graph shows the number of eggs laid by 5 animals in a 2.5 hour period. eipr-1 mutants show egg-laying defects and this phenotype is rescued by panneuronal expression of either the worm gene or its mouse ortholog . Error bars = SEM; n = 10 plates of 5 worms each. (C) eipr-1 acts in the same pathway as rab-2 to control egg-laying. Double mutants of eipr-1(tm4790) with rab-2(nu415) or rund-1(tm3622) do not have stronger egg-laying defects than the single mutants. Though rund-1(tm3622) mutants have a visible Egl (egg-laying defective) phenotype indicating that they retain more eggs, they did not have a significantly reduced egg-laying rate as measured by this assay. Error bars = SEM; n = 5 plates of 5 worms each. ns, not significant, P>0.05.(A) The activated Gq mutant (TIF)Click here for additional data file.S2 FigC. elegans EIPR-1  and its orthologs from Arabidopsis thaliana , Drosophila melanogaster , and Homo sapiens . Identical residues are shaded in black and similar residues are shaded in gray. The WD40 repeats  are marked with single black bars. Using SMART, worm EIPR-1 has four predicted WD40 repeats, Arabidopsis has six, fly has three, and human has five. WD40 repeats are difficult to identify by primary sequence and are often missed by prediction programs. The position of the ox316 stop mutation is marked with an asterisk. Alignment was made with MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/) using default parameters and exhibited with Boxshade 3.21 (http://www.ch.embnet.org/software/BOX_form.html).Alignment of (TIF)Click here for additional data file.S3 FigceIs61) fluorescence in dorsal cord motor neuron axons of the wild type and eipr-1(tm4790) mutant strains. Scale bar: 10 μm. eipr-1(tm4790) mutants have decreased fluorescence in the dorsal cord, indicative of a FLP-3::Venus sorting or trafficking defect. (B) Representative images of INS-22::Venus (nuIs195) fluorescence in dorsal cord motor neuron axons. Scale bar: 10 μm. (C) Representative images of IDA-1::GFP (ceIs72) fluorescence in the dorsal nerve cord. Scale bar: 10 μm. (D) Levels of the synaptic vesicle cargo synaptobrevin, SNB-1::GFP (nuIs152), in the dorsal nerve cord. eipr-1(tm4790) mutants do not have a defect . Error bars = SEM; n = 21–26. (E) eipr-1 and vps-50 mutants do not have enlarged lysosomes. Representative images of LMP-1::GFP (pwIs50) fluorescence in coelomocytes of wild-type (WT), eipr-1(tm4790) and vps-50(ok2627) mutant strains. Scale bar: 10 μm.(A) Representative images of FLP-3::Venus ((TIF)Click here for additional data file.S4 Figeipr-1 is located. The image on the left shows expression in the hypodermis (skin) while the image on the right shows expression in the pharynx. (B) Representative images of neurons expressing eipr-1::GFP under panneuronal  and head cholinergic  promoters. Scale bars: 5 μm. (C) Representative images of neurons coexpressing rab-3p::eipr-1::GFP and GTP-bound RAB-2 (RAB-2QL). Scale bars: 5 μm. (D) Representative images of 832/13 cells expressing GFP::RAB2A and costained for endogenous VPS50. Scale bar: 10 μm. VPS50 partially colocalizes with RAB2A. (E) Representative images of 832/13 cells expressing CCCP1::GFP and costained for endogenous VPS50. Scale bar: 10 μm. VPS50 largely colocalizes with CCCP1. (F) Representative images of wild-type (WT) and eipr-1(tm4790) mutant neurons expressing VPS-52::tagRFP. Scale bars: 1 μm. VPS-52 localizes normally in eipr-1 mutants.(A) Representative images of animals expressing GFP under the promoter region upstream of the first gene (W09C5.1) of the operon where (TIF)Click here for additional data file.S5 Fig(A) List of top hits from two independent experiments performing mass spectrometry of a pulldown of rEIPR1::GFP in 832/13 cells. On the left side of the table, we show the list of hits found in a mass spec experiment after subtracting hits found in a GFP control pulldown. On the right side of the table, we show the list of hits found in an independent mass spec experiment after subtracting hits found in either the pulldown of CCCP1::GFP or RUNDC1::GFP. In both cases, EIPR1::GFP pulldowns were performed in parallel to controls. # seq = number of unique peptides from each protein. All proteins with two or more unique peptides are shown. (B) EGFP-tagged rat EIPR1 or EGFP was expressed in 832/13 cells. Immunoprecipitation of EIPR1::EGFP pulled down more SNAP29 than untagged EGFP. IN: input; IP: immunoprecipitation.(TIF)Click here for additional data file.S6 Figvps-50 acts in the same genetic pathway as eipr-1 to control FLP-3::Venus trafficking. Left: representative images. Right: quantification. vps-50(ok2627) and eipr-1(tm4790) have reduced FLP-3::Venus (ceIs61) fluorescence, but an eipr-1(tm4790); vps-50(ok2627) double mutant does not have a stronger phenotype than either single mutant indicating that eipr-1 and vps-50 act in the same genetic pathway. A vps-54(ok1473) mutant does not have decreased axonal levels of FLP-3::Venus. ***, P<0.001 compared to wild type. Error bars = SEM; n = 9–14. (B) eipr-1 and GARP/EARP mutants have reduced secretion of NLP-21::Venus. NLP-21::Venus (nuIs183) fluorescence levels in the coelomocytes of the indicated strains. Like rab-2(nu415) mutants, eipr-1(tm4790), vps-50(ok2627), and vps-52(ox345) mutants show reductions in the accumulation of Venus fluorescence in coelomocytes that is approximately proportional to the decrease in axonal fluorescence seen in these mutants, suggesting that these mutants are not defective in dense-core vesicle release. Double mutants between eipr-1(tm4790) and rab-2(nu415) or vps-50(ok2627) do not have stronger phenotypes than the single mutants, suggesting that these genes all act in the same dense-core vesicle cargo sorting pathway. **, P<0.01; ***, P<0.001 compared to WT. Error bars = SEM; n = 14–33. (C) vps-51 mutants have defects in trafficking FLP-3::Venus. FLP-3::Venus (ceIs61) fluorescence levels in the dorsal nerve cord of wild type and vps-51(tm4275) mutant strains. Error bars = SEM; n = 8. (D) GARP mutants vps-54(ok1463) and vps-54(ok1473) do not have a reduced locomotion rate. Error bars = SEM; n = 10. (E) eipr-1(tm4790) and vps-50(ok2627) mutants do not have defects in trafficking synaptic vesicle cargos. SNB-1::mCherry (ceIs61) fluorescence levels were measured in the dorsal nerve cord. ns, not significant, P>0.05 compared to wild type. Error bars = SEM; n = 11–14.(A) (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.


We propose and evaluate a new combined fat-water separated saturation-recovery imaging sequence (IDEAL-T1) for water-separated T1 mapping.Myocardial T1 approach combines a gated, segmented multi-echo gradient recalled echo readout for fat-water separation, based on the "iterative decomposition of water and fat with echo asymmetry and least squares estimation" (IDEAL) method[1 mapping[1 and T2 values (14 phantoms), IDEAL-T1 was validated against an inversion spin-echo sequence. In-vivo evaluation of myocardial T1 was completed in 6 healthy individuals and compared to a single-shot saturation recovery sequence (SASHA)[The IDEAL-T1 mapping-4. Image1, T2, and off-resonance (up to 250 Hz), but dependence on B1 errors and saturation efficiency. Phantom experiments show excellent correlation with spin-echo values  with a mean underestimation of 2.4 ms (Figure Simulations reveal negligible dependence on T1 provides the benefit of fat-water separation with quantitative myocardial T1-mapping with a small underestimation in T1. Areas of thin myocardium, including the right ventricle, may benefit from resolving zones of partial voluming with fat by having water only images for analysis.IDEAL-TThe authors acknowledge financial support from CIHR, AIHS, WCHRI.


Escherichia coli impedes the biological synthesis of polyketides, flavonoids and biofuels. Here, a clustered regularly interspaced short palindromic repeats (CRISPR) interference system was constructed for fine-tuning central metabolic pathways to efficiently channel carbon flux toward malonyl-CoA. Using synthetic sgRNA to silence candidate genes, genes that could increase the intracellular malonyl-CoA level by over 223% were used as target genes. The efficiencies of repression of these genes were tuned to achieve appropriate levels so that the intracellular malonyl-CoA level was enhanced without significantly altering final biomass accumulation . Based on the results, multiple gene repressing was successful in approaching the limit of the amount of malonyl-CoA needed to produce the plant-specific secondary metabolite (2S)-naringenin. By coupling the genetic modifications to cell growth, the combined effects of these genetic perturbations increased the final (2S)-naringenin titer to 421.6 mg/L, which was 7.4-fold higher than the control strain. The strategy described here could be used to characterize genes that are essential for cell growth and to develop E. coli as a well-organized cell factory for producing other important products that require malonyl-CoA as a precursor.The limited supply of intracellular malonyl-CoA in  Escherichia coli and Saccharomyces cerevisiae, the endogenous central metabolism predominates and strongly competes for carbon sources and energy during malonyl-CoA synthesis, leaving only small amounts available for the production of recombinant products15615Malonyl-coenzyme A  is an important precursor metabolite involved in the biosynthesis of many important chemicals, such as flavanones3Photorhabdus luminescensRhizobium trifolii10sdhA (succinate dehydrogenase), adhE , brnQ (branched chain amino acid transporter) and citE (citrate lyase)fumC (fumarate hydratase) and sucC (succinyl-CoA synthetase)For example, overexpression of four acetyl-CoA carboxylase (ACC) subunits and biotin ligase from The use of RNA-mediated regulatory mechanisms for fine flux control has been well documented1316E. coli requires 3 mol of malonyl-CoA, which imposes a significant metabolic burden on the host strain15Flavonoids are valuable natural products widely used in human health and nutrition due to their biochemical properties, which include antiviral, anti-obesity and anti-cancer activities18600 decreased by less than 10%). Finally, multiple gene repressing was performed to achieve a high yield of (2S)-naringenin (421.6 mg/L). This strategy enhances the malonyl-CoA concentration without the need to add substrates for malonyl-CoA generation, which potentially provides an economically sustainable process for the efficient production of other plant natural compounds.Here, we explored the impact of fine-tuning the central metabolic pathways by using a CRISPRi systemE. coli, a catalytically dead Cas9 mutant (dCas9) derived from Streptococcus pyogenes cas9 geneT7 promoter. The sgRNA molecule, which consists of four domains  complementary region for specific DNA binding, a 42-nt dCas9-binding hairpin and a 40-nt transcription terminator derived from S. pyogenes), was coexpressed with dCas9To implement the CRISPRi platform in zwf (glucose-6-phosphate 1-dehydrogenase), pgl (6-phosphogluconolactonase), tpiA (triosephosphate isomerase), ppsA (phosphoenolpyruvate synthase), eno (phosphopyruvate hydratase), glyA (serine hydroxymethyltransferase) and fold  were chosen as target genes. Consumption of acetyl-CoA in other central metabolic pathways, such as the TCA cycle and glycolysis, needs to be reduced and hence, mdh , fumC, sdhABCD (succinate dehydrogenase), sucCD (succinyl-CoA synthetase), sucA (2-oxoglutarate dehydrogenase), sucB (dihydrolipoamide acetyltransferase), acnAB (aconitate hydratase), citE (citrate lyase), gdhA (glutamate dehydrogenase), gltp (glutamate/aspartate:proton symporter) and adhE  were also chosen (fabH (3-oxoacyl-acyl carrier protein synthase III), fabB (3-oxoacyl-acyl carrier protein synthase I), fabF (3-oxoacyl-acyl carrier protein synthase II), fabG (3-oxoacyl-acyl carrier protein reductase), fabA (3-hydroxydecanoyl-acyl carrier protein dehydratase), fabI (enoyl-acyl carrier protein reductase) and fabD  were chosen as target genes , while sgRNAs targeting ppsA, eno, adhE, mdh, fumC, sdhA, sucC and citE produced a dramatic increase in malonyl-CoA concentration (over 223%). It was also found that sgRNAs targeting fabH, fabB, fabF and fabI produced a simultaneous increase in acetyl-CoA and malonyl-CoA concentration (over 244%)  . Hence, Acetyl-CoA and malonyl-CoA are important metabolic intermediates involved in central metabolic pathways, such as glycolysis, the TCA cycle422fabF increased the malonyl-CoA concentration by 433.3% and decreased the final biomass by 9% , which yielded a production titer of 50.5 mg/L. Appropriate repressing efficacy toward a target gene that could increase the malonyl-CoA concentration without significantly altering the final biomass was ranked as a beneficial genetic perturbation. The impacts of these independent genetic interventions, namely low repressing efficacy toward eno, adhE, mdh and fabB, medium repressing efficacy toward sucC and fumC and high repressing efficacy toward fabF, on (2S)-naringenin production were analyzed. Single perturbations of eno, adhE, mdh, fabB, fabF, sucC and fumC increased production by up to 38.6%, 74.3%, 86.1%, 54.5%, 135.6%, 78.2% and 96.1%, respectively, and perturbation of fabF resulted in the highest (2S)-naringenin production (119.6 mg/L) (Based on a previous study.6 mg/L) .fabF sgRNA in combination with anti-eno, anti-adhE, anti-fabB, anti-sucC or anti-fumC sgRNA but not anti-mdh produced a higher (2S)-naringenin titer than single anti-fabF sgRNA. The latter result is probably due to an imbalance between cell growth and product formation because it was observed that the strain anti-fabF/mdh resulted in the lowest cell growth (fabF/fumC sgRNA produced the highest (2S)-naringenin titer (220.1 mg/L).The impacts of different synthetic sgRNA combinations on (2S)-naringenin production were evaluated. Firstly, the effect of two genetic perturbations on (2S)-naringenin production was investigated. Anti-l growth . Among tfabF/fumC sgRNA in combination with anti-eno, anti-adhE, anti-fabB or anti-sucC sgRNA was investigated. Each sgRNA combination produced a higher (2S)-naringenin titer than anti-fabF/fumC, and anti-fabF/fumC/fabB produced the highest (2S)-naringenin titer (298.8 mg/L) -naringenin titer than anti-fabF/fumC/fabB sgRNA, and anti-fabF/fumC/fabB/sucC sgRNA produced the highest (2S)-naringenin titer (398.3 mg/L). Anti-fabF/fumC/fabB/sucC sgRNA in combination with anti-eno, or anti-adhE sgRNA produced a higher (2S)-naringenin titer than anti-fabF/fumC/fabB/sucC sgRNA, and anti-fabF/fumC/fabB/sucC/adhE sgRNA produced the highest (2S)-naringenin titer (421.6 mg/L). However, anti-fabF/fumC/fabB/sucC/adhE/eno sgRNA produced a lower (2S)-naringenin titer (384.5 mg/L) than anti-fabF/fumC/fabB/sucC/adhE sgRNA  . SimilarhE sgRNA .Identification of potential genetic targets and optimization of their expression are essential for the efficient production of desired metabolites15fabB and fabF to enhance the intracellular malonyl-CoA concentrationThere has been increasing interest in the use of RNA-mediated regulatory mechanisms for target gene regulation16Photorhabdus luminescens and biotin ligase from the same speciesRhizobium trifolii10Previous studies have made significant gains in improving the intracellular malonyl-CoA concentration and achieved a high yield of flavonoids through: 1) the ACC route, which requires coordinated overexpression of four acetyl-CoA carboxylase (ACC) subunits from  the ACC mdh which resulted in the lowest cell growth, the combined effects of other genetic modifications on production were additive  was used to express sgRNA and produce flavonoids. The compatible vector set  was used to express multiple genes in one strain 4Cl was used for sgRNA expression and flavonoid production. Ampicillin (100 μg/mL), kanamycin (40 μg/mL), chloramphenicol (20 μg/mL), and streptomycin (40 μg/mL) was added when required. Cell growth was monitored by measuring the absorbance at 600 nm (OD600) with a UV/Vis spectrophotometer . The primers and plasmids used in this study are listed in i BL21 DE was usedNcoI/HindIII sites of pACYCDuet-1. The sgRNA chimera, which consists of four domains  complementary region for specific DNA binding, a 42-nt dCas9-binding hairpin and a 40-nt transcription terminator derived from Streptococcus pyogenespfoI/BamHI sites of pCOLADuet-1 Trc promoter, 20-bp complementary region, 42-bp dCas9-binding hairpin and 40-bp transcription terminator were cloned into the same plasmid. The primer pair Pf_sgRNA(BamHI)/Pr_sgRNA(EcoRI) was used to amplify anti-adhE, anti-eno, anti-mdh, anti-fabB, anti-fumC and anti-sucC sgRNA sequences into the BamHI/EcoRI sites of pCOLA-fabF(high). This resulted in the plasmids pCOLA-fabF(high)/adhE(low), pCOLA-fabF(high)/eno(low), pCOLA-fabF(high)/mdh(low), pCOLA-fabF(high)/fabB(low), pCOLA-fabF(high)/fumC(medium) and pCOLA-fabF(high)/sucC(medium). The primer pair Pf_sgRNA(EcoRI)/Pr_sgRNA(HindIII) was used to amplify anti-adhE, anti-eno, anti-fabB and anti-sucC sgRNA sequences into the EcoRI/HindIII sites of pCOLA-fabF(high)/fumC(medium). This resulted in the plasmids pCOLA-fabF(high)/fumC(medium)/adhE(low), pCOLA-fabF(high)/fumC(medium)/eno(low), pCOLA-fabF(high)/fumC(medium)/fabB(low) and pCOLA-fabF(high)/fumC(medium)/sucC(medium). The primer pair Pf_sgRNA(HindIII)/Pr_sgRNA(NdeI) was used to amplify anti-adhE, anti-eno and anti-sucC sgRNA sequences into the HindIII/NdeI sites of pCOLA-fabF(high)/fumC(medium)/fabB(low). This resulted in the plasmids pCOLA-fabF(high)/fumC(medium)/fabB(low)/adhE(low), pCOLA-fabF(high)/fumC(medium)/fabB(low)/eno(low) and pCOLA-fabF(high)/fumC(medium)/fabB(low)/sucC(medium). The primer pair Pf_sgRNA(NdeI)/Pr_sgRNA(BglII) was used to amplify anti-adhE and anti-eno sgRNA sequences into the NdeI/BglII sites of pCOLA-fabF(high)/fumC(medium)/fabB(low)/sucC(medium). This resulted in the plasmids pCOLA-fabF(high)/fumC(medium)/fabB(low)/sucC(medium)/adhE(low) and pCOLA-fabF(high)/fumC(medium)/fabB(low)/sucC(medium)/eno(low). The primer pair Pf_sgRNA(BglII)/Pr_sgRNA(KpnI) was used to amplify anti-eno sgRNA sequences into the BglII/KpnI sites of pCOLA-fabF(high)/fumC(medium)/fabB(low)/sucC(medium)/adhE(low). This resulted in the plasmid pCOLA-fabF(high)/fumC(medium)/fabB(low)/sucC(medium)/adhE(low)/eno(low).To repress multiple genes, different sgRNA sequences including the 600 of 0.1. After the OD600 reached 1.65, an additional 25 mL of fresh MOPS medium was added. sgRNA expression was induced with 0.5 mM IPTG. Cultures were subsequently incubated at 30 °C. 1 mL of cell culture was harvested at the mid-log phase of growth to quantify the intracellular concentrations of malonyl-CoA and acetyl-CoA. Final OD600 values of the cultures were measured after a total fermentation time of 48 h.To investigate the silencing effects of various sgRNA species, cells were cultured in 25 mL of MOPS medium at 37 °C with 220 rpm orbital shaking at a starting OD600 of 0.1. After an OD600 of 1.65 had been reached, an additional 25 mL of fresh MOPS medium was added. Cultures were subsequently incubated at 30 °C for (2S)-naringenin production. IPTG and L-tyrosine were provided at final concentrations of 1 mM and 3 mM, respectively. After a total fermentation time of 48 h, 1 mL of cell culture was collected to measure the concentrations of (2S)-naringenin and p-coumaric acid600 values of the cultures were also measured after a total fermentation time of 48 h.For (2S)-naringenin production, cells were first cultured in 25 mL of MOPS medium at 37 °C with 220 rpm orbital shaking at a starting ODp-coumaric acid production, E. coli cells were separated through centrifugation . 1 mL of supernatant was extracted with an equal volume of ethyl acetate. After vortexing and centrifugation , the top organic layer was separated and evaporated to dryness, and the remaining residue was resolubilized with 1 mL of methanol. Samples were analyzed by high-performance liquid chromatography (HPLC), using an Agilent 1100 series instrument and a reverse-phase Gemini NX-C18 column (5 × 110 mm) maintained at 25 °C. (2S)-Naringenin and p-coumaric acid were separated by elution with an acetonitrile/water gradient at a flow rate of 1.0 mL/min under the following conditions: 10 to 40% acetonitrile (vol/vol) for 10 min, 40% acetonitrile (vol/vol) for 5 min, 40 to 10% acetonitrile (vol/vol) for 2 minFor each experiment, triplicate cultures were measured, and their deviation is represented by the error bar. To analyze (2S)-naringenin and g and 4 °C for 10 min. The cell pellet was resuspended in 1 mL of 6% perchloric acid to facilitate cell lysis. The lysed cell suspension was then neutralized with 0.3 mL of 3 M potassium carbonate while vortexing to neutralize the acid. The solution was centrifuged to pellet the cell debris. The supernatant was collected and stored chilled until the malonyl-CoA content was analyzed. To determine the dry cell weight, 2 mL of the same culture were filtered through a 0.45-μm cellulose membrane , followed by washing with distilled water and drying in a conventional oven. Dry cell weight was represented by the weight difference between empty membranes and those with cell residuesTo quantify the malonyl-CoA concentration, an aliquot of 1 mL of cell culture was removed and chilled immediately on ice and centrifuged at 5000 −). A liquid chromatography-mass spectrophotometer  equipped with an electrospray ionization (ESI) source and a reverse-phase Gemini NX-C18 column (5 × 110 mm) was used. A Shim-Pack VP-ODS 150L × 2.0 HPLC column  was used to perform HPLC separation. Samples of malonyl-CoA and acetyl-CoA were eluted at a flow rate of 0.3 mL/min with a gradient of 15 mM ammonium formate (A) and 10% 10 mM ammonium acetate in methanol (v/v) (B) as follows: at 0 min, 2% B; 10 min, 60% B; 20 min, 76% B; 25 min, 2% B. The MS/MS system was operated in negative ion mode using optimized conditions: detector voltage, 1.60 Kv; nebulizing gas (N2) flow, 1.5 L/min; drying gas (N2) flow, 200 kPa; ion accumulation time, 30 ms; scan range m/z, 100–1000 for MS1, 100–500 for MS2.Malonyl-CoA and acetyl-CoA from the cell cultures were identified by the area of major mass spectra signals .


Drosophila neuromuscular junction(NMJ) depend on trans-synaptic neurexin/neuroligin signalling, which is promoted bythe scaffolding protein Syd-1 binding to neurexin. Here we report that the scaffoldprotein spinophilin binds to the C-terminal portion of neurexin and is needed tolimit neurexin/neuroligin signalling by acting antagonistic to Syd-1. Loss ofpresynaptic spinophilin results in the formation of excess, but atypically smallactive zones. Neuroligin-1/neurexin-1/Syd-1 levels are increased atspinophilin mutant NMJs, and removal of single copies of theneurexin-1, Syd-1 or neuroligin-1 genes suppresses thespinophilin-active zone phenotype. Evoked transmission is strongly reduced atspinophilin terminals, owing to a severely reduced release probability atindividual active zones. We conclude that presynaptic spinophilin fine-tunesneurexin/neuroligin signalling to control active zone number and functionality,thereby optimizing them for action potential-induced exocytosis.Assembly and maturation of synapses at the  Synaptic assembly depends on trans-synaptic Neurexin/Neuroliginsignalling. Here, Muhammad et al. show that Spinophilin, a pre-synapticscaffolding protein, interacts with Neurexin, in competition with Syd-1, to regulate theformation and function of synaptic active zones at Drosophila neuromuscularjunctions. Drosophila homologue is calledBruchpilot (BRP))156in vitro, synapses ofrodents8Caenorhabditis elegansDrosophila1112131415C. elegansDrosophilaDrosophila, the Syd-1-PDZ domain binds the Nrx-1 C terminus and couples pre-with postsynaptic maturation at nascent synapses of glutamatergic neuromuscularjunctions (NMJs) in Drosophila larvae. Syd-1 cooperates with Nrx-1/Nlg1 tostabilize newly formed AZ scaffolds, allowing them to overcome a‘threshold' for synapse formationSpn NMJ terminals.Chemical synapses release synaptic vesicles (SVs) at specialized presynaptic membranes,so-called active zones (AZs), which are characterized by electron-dense structures,reflecting the presence of extended molecular protein scaffolds. These AZ scaffoldsconfer stability and facilitate SV release23Drosophila larvae continuously expand to meet therequirements of the growing muscle fibres by adding new release sites (orsynapses) to their structure19Drosophila NMJ model system tosearch for factors restricting the number of BRP scaffolds. To this end, a setof proteins and their known binding partners, which we previously detected inimmunoprecipitation experiments against BRPDrosophila homologue of theNeurabin/Spn family caused an increase of AZ numbers at the NMJ  . Neurabin/Spn family proteins in rodents are stronglyexpressed in postsynaptic spines2426Drosophila NMJs. To validate this hypothesis, and to determine Spnlocalization, we raised a polyclonal antibody against a fusion protein from theSpn N-terminal region ), proving the specificity of the NMJ Spnantibody signal (horseradish peroxidase (HRP) signal, which outlinesthe neuronal membrane  . Genomict larvae . Staininy signal . To charmembrane . Muscle-membrane remainedSpn) we created  labellingspn locus (Pac(Spn1&2); Spn null phenotype. Both genomic constructs fullyrescued adult viability and, importantly, the NMJ phenotypes of Spn. Inaddition, deletion of a stretch encoding the Spn open reading frame within thegenomic construct of Pac(Spn2), named Pac(Spn*),abolished rescue activity (data not shown). We further tested a semi-lethaltransposon insertion within the spn locus(Mi(Mic)SpnMI06873), which we found to significantly reduceanti-Spn staining. The latter mutant showed NMJ phenotypes similar, but somewhatweaker, than those observed in Spn null larvae  were significantly increased in Spn when compared withcontrols (elav(x)-C155-gal4 in the Spn null backgroundto test whether this was due to a loss of presynaptic Spn. Indeed, presynapticexpression of Spn complementary DNA (cDNA) effectively re-established normal AZdensities  is not sufficient to reliablyquantify AZ scaffold size. Thus, we turned to stimulated emission depletion(STED) microscopy operating with ≈45 nm lateralresolution21Spn mutants, while presynapticSpn re-expression restored normal AZ size  analysis consistentlyrevealed smaller but otherwise normal T-bars . In someSpn. As details of the GluRorganization may not be resolved by standard confocal imaging, we usedthree-dimensional structured illumination microscopy (3D SIM) with an isotropicresolution of ≈120 nmz-axis, whileSTED only increases the x–y resolution. Therefore, SIM allowedus to resolve the 3D organization of GluR fields relative to the AZs. Consistentwith the EM analysis, Spn NMJs showed extended, often continuous receptorfields, juxtaposed to several small AZs, with a clear increase in the area ofthe postsynaptic compartment labelled with GluRs , which show fewer but larger and oftenmis-shapen AZ scaffolds1315Spn terminals, using an antibodydetecting endogenous Nrx-1  analysis showed elevated mobility ofNrx-1GFP in a Syd-1 mutant backgroundSpn were upregulated , another scaffolding proteinthat binds to the Nrx-1 C terminus36GFP clusters appeared to be unchanged at Spnterminals (even though the cluster density was increased)  . Thus,SGFP being recruited into AZsGFP andmStrawberrySyd-1, both the Nrx-1GFP level andmStrawberrySyd-1 level at AZs dropped .mStrawSyd-1 intensity in wild-type background:1.0±0.04, n=20; mStrawSyd-1intensity in the presence of Spn: 0.76±0.05; P<0.01;Mann–Whitney t-test (U=75)). Thus, Spngain-of-function might influence Nrx-1, antagonistic to the Spn loss-of-functionphenotype .We further investigated whether, as suggested by the Nrx-1 FRAP data, Syd-1 andSpn would operate in a competitive manner. Consequently, we revisited ourprevious finding that overexpression of Syd-1 within motoneurons results inco-expressed Nrx-1henotype . In factDrosophila headextractsDrosophila and rodents  no longer rescued the structuralpresynaptic AZ phenotype of Spn mutants. As expected, expression ofwild-type cDNA  was re-expressed in the presynaptic motoneuron  to assay SV release.We observed a clear increase in the frequency of spontaneous SV release fromtoneuron . Howeverned high .The amperminals , potentierminals . In contSpn NMJs. Loss oflitation . Both deSpn NMJs, suggesting thatthe net increase of spontaneous events observed in TEVC experiments is, indeed,due to an increase in synapse number rather than in their individual releaserates correlated with BRP level. However, as this relationship differed from thatobserved in control animals we can rule out the possibility that the effect ismediated solely through BRP reduction. Thus, we conclude that Spn is not onlyimportant for controlling synapse number and size, but also for optimizingaction-potential-induced exocytosis by enhancing release probability atindividual AZs.The TEVC recordings sample release events over the whole NMJ of the respectivemuscle, but do not allow for the analysis of individual AZs. To investigate thelatter, we used a recently developed assay employing post-synaptically expressedGCaMP to characterize the spatial and temporal dynamics of exocytoticevents23serates . By contserates . Howeverrol NMJs . ConsistMJs .The trans-synaptic dialogue between Nrx-1 and Nlg1 aids in the initial assembly,specification and maturation of synapses, and is a key component in the modificationof neuronal networks1241C. elegans identified roles of Syd-1epistatic to Syd-2/Liprin-α in synaptogenesis5Drosophila NMJs174921nrx-1, nlg1 or syd-1, Spn displays all thefeatures of a ‘negative' element mounting, which effectivelysets the threshold for AZ assembly. As suggested by our FRAP experiments , one of the two Neurabin protein families presentin mammals, was reported to provoke a developmental increase in synapse numbers26434445eriments , Spn migeriments . As a neC. elegans Spn homologue NAB-1 (NeurABin1) was previously shown tobind Syd-1 in cell culture recruitment assaysC. elegans HSN synapses, a previous studyetal.Drosophila larval NMJs) where an already functional neuronalterminal adds novel AZs17Drosophila NMJs. F-actin patches might be particularly important toestablish the first synaptic contacts between partner cells. Both the study by Chiaet al. and this study, however, point clearly towards importantregulatory roles of Spn family members in the presynaptic control of synapseassembly.The S6K . It will be interesting to see whether thisinteraction can be differentially regulated, for example, by(de)phosphorylation.Further, we describe a novel interaction between the Spn-PDZ domain and theintracellular C-term of Nrx-1 at the atomic level. Interestingly, we found that allfunctions of Spn reported in this study, structural as well as functional, werestrictly dependent on the ligand-binding integrity of this PDZ domain. It isnoteworthy that the Spn-PDZ domain binds other ligands as well, for example,Kalirin-7 and p70S6K refs , 54, 55,eriments . NonetheDrosophilaNMJsGFP motility using FRAP, genetic ablation of CASK had noeffect  wasvalidated by genomic PCRspnΔ3.1in trans with the deficiency chromosome dfBSc116 (Spn deficiency: Df)resulted in animals deficient in the Spn locus. Lethality in Spn wascompletely rescued by returning one copy of the genomic region of Spn in thismutant background. It is of note that another mutant allele of Spn wasreported previously and was shown to be ‘semi-lethal'1118w served as a genetic background for allexperiments. Recombinations were verified using PCR or complementation analysis.The following recombination lines were used: forSyd-1, Nrx-1 andNlg1. Flies carryingUAS–green fluorescent protein (GFP)-tagged Nrx-1 was constructed by circular PCR using primers: 5′-GTGGAATTGATGGCGGGTCCTGAGGGTGCGGGTCTCAGTATAATTG -3′ and 5′-CAATTATACTGAGACCCGCACC CTCAGGACCCGCCATCAA TTCCAC -3′.The point-mutated PDZ domain of Spn cDNA , dmSyd-1 (residue 155–242) andrnSpn (residue 493–583) were amplified by PCR and cloned intothe pET-MBP vector using NcoI and SalI restriction sites with primers:dmSpn_fwd: 5′- TATACCATGGCGCATGTCTTCCCCGTGG -3′,dmSpn_rev: 5′- TATA CCATGGTGGCCGCTTCGG -3′,dmSyd-1_fwd:5′- TATACCATGGCGCAGGCGGTCGATGC -3′,dmSyd-1_rev:5′- TATACCATGGCGCACACGGTTCAACTTGTCG-3′, rnSpn_fwd: 5-′ TATACCATGGAGCTGTTTCCTGTGGAG-3′ and rnSpn_rev: 5′- ATATGTCGACCTACTCCCGGCCAATCATG-3′.The constructs comprising the PDZ domains of dmNrx-1 ct_rev: 5′-TTACACATACCACTCCTTGACGTC CTTGG AGTC GTCACGATGCGGCC -3′,rNrx-ct_fwd: 5′- AAGAAGAACAAAGACAAAGAGTATTACGTCTAGCTGACGATCTGCCTCG -3′, rNrx-1ct_rev: 5′-CTAGACGTAATACTCTTTGTCTTTGTTCTTCTTGTCAC GA TGCGGCC-3′.The resulting constructs contained an N-terminal His6-MBP-tag followed by atobacco etch virus cleavage site and the respective PDZ domain. The constructscomprising the last 10 C-terminal amino acids of dmNrx-1  and rnNrx-1  wereamplified by PCR and cloned into the pGEX-6-P1 vector by a SLIC reaction usingoverlapping primers: dmNrx-1ct_fwd: 5′- GACTCCAAGGACGTCAAGGAGTGGTATGTGTAACTGACGATCTGCCTCG -3′, The resulting constructs comprised an N-terminal GST-tag followed by aPreScission cleavage site and the respective 10 C-terminal amino acids of Nrx-1.Detailed version of methods for Protein expression and purification, ITC assaysand crystallization are presented in 1) was created from P[acman] BAC cloneCH321-01N11 , which was subjected to transgenesis using the Phi31 system.Similarly, Pac(Spn2) was obtained by injecting theP[acman] BAC clone CH321-67O06 (genomic region 2469714 to2556468). Pac(Spn*) corresponds to P[acman] BAC cloneCH321-67O06, but lacks the whole Spn open reading frame, and was clonedaccording to the Counter Selection BAC Modification kit obtained from GeneBridges GmbH. rpsL-neomycin (neo) template DNA was used to generate selectablecassettes. Primers contained a 50-bp homology region and a sequence foramplification of the rpsL-neo counter selection cassette. Selectable cassetteswere generated by PCR using Vent Polymerase  and thefollowing primer pairs. Spn-rpsL-fwd:5′-GGCCCGAAATTCAAGCTAAACGGACGCGTTTTCGTCGCGAGTTTAACC GCGGCCTGGTGATGATGGCGGGATCG-3′, Spn-rpsL-rev: 5′-ATTTCAGAGTATATTTATTAGCACTGATTTTGAGATTTATT ATTTTCCATTCAGAAGAACTCGTCAAGAAGGCG-3′.Pac . The cytoplasmic C terminus of Nrx-1 was cloned into pB27 usingprimers: 5′- GATGGAATTC-AATGGCGATCGTGGCT -3′ and5′- GTCTATACTAGT-TTACACATACCACTCCTTGACGTCCT -3′.The Spn and Syd-1 fragments depicted in N2.2 , RbGluRIID (1:500), RbDSyd-1 (1:500),RbNlg1 (1:500), RbDRBP (1:500) and guinea pig Nrx-1 (1:500) (generously providedby M. Bhat). We used MNc82 (1:100) and MCSP (1:500) , the University of Iowa, Iowa City, IA), MFasII , mouse monoclonal antibody 3E6 (to stain GFP) (1:500) (Invitrogen) andrabbit anti-dsRed (1:500) (Clontech). Secondary antibodies were generallydiluted 1:500. Secondary antibodies for STED were used in the followingconcentrations: goat anti-mouse Atto590 1:100 and goat anti-rabbit star6351:100. The dyes Atto590 (ATTO-TEC) and Star635 (Abberior) were coupled to thestated IgGs (Dianova). Imaging larvae were mounted in Mowiol (Sigma-Aldrich) forSTED.Larval filets were dissected and stained as described previously13nc82, RimBP and CacGFP) were quantifiedfrom maximal projections of confocal NMJ stacks. A Cy5-HRP antibody  was used to outline the shape of the NMJ. Controland mutant larvae were stained in the same vial. All images for synapsequantification from fixed samples were acquired using the same microscopesettings . AZ cluster analysis was done as described previouslyThe sizes and surface densities of AZ cluster . Membranes were probed withguinea pig anti-Nrx-1  and rabbit anti-SpnN2.2 diluted in PBS-T. After washing, secondary anti-guinea pig or anti-rabbitHRP-conjugated antibodies were used for detection (Dianova) in conjunction withan enhanced chemoluminescence detection system with Hyperfilm ECL . Films were scanned intransmission mode (Epson V770). Images were imported to Photoshop (Adobe), andbrightness and contrast were adjusted. The liquid Y2Hβ-galactosidase assay was performed as reported previouslyWe followed a new protocol using  Epson V7. Images g for30 min. About 10 μl per ml homogenate ofprotein A/G magnetic beads were added following 30 min incubation andseparation of magnetic beads from the homogenate. Homogenate was aliquoted in2 ml tubes (1.6 ml per tube) and0.8 μg affinity-purified anti-pan-NRX or rabbit IgG wasadded to each aliquot. Samples were incubated overnight with rotation at4 °C. About 8 μl protein-A magneticbeads (Dynabeads) were added and samples were incubated for additional2 h. Samples were washed 3 × with homogenizationbuffer+0.1% Triton-X100 and once with homogenization bufferwithout detergent. Bound proteins were eluted with 30 μl2% sodium deoxycholate. Eluted proteins were separated on8% PAA gel and probed with anti-spinophilin  and anti-pan-Nrx.Brains were homogenized in 25 ml per g tissue in homogenization buffer using glass homogenizer. After homogenization samples weresonicated with 3 × 10 pulses, Triton-X100 was added to the finalconcentration of 1% and homogenate was incubated for10 min at 4 °C with rotation. Sample was sonicatedagain with 10 pulses. Samples were spun down at 20,000 × 3 10, trehalose 5, sucrose 115, HEPES 5 and CaCl2 1.5,pH adjusted to 7.2. Recordings were made from cells with an initial membranepotential (Vm) between −50 and −70 mV andinput resistances of ⩾4 MΩ, using intracellularelectrodes with resistances of 8–20 MΩ and filledwith 3 M KCl. eEJCs, which reflect the compound excitatory junctionalcurrent of both the motoneurons innervating muscle 6 (voltage clamp at−60 mV) and mEJCs (voltage clamp at−80 mV) were low pass filtered at 1 kHz. The0.2-Hz stimulation protocols included 20 traces per cell. Paired-pulserecordings consisted of 10 traces per interval per cell in which a 4-s rest wasleft between paired pulses. For determination of the base line of the secondpulse at the 10-ms interpulse interval, the decay of the first pulse wasextrapolated. Recordings were analysed with pClamp 10 (Molecular Devices).Stimulation artifacts in eEJC recordings were removed for clarity.TEVC recordings were performed on larval NMJs of third instar males (muscle 6 andsegments A2 and A3), essentially as described2+ imaging,image alignment and signal processing.Optical analysis of spontaneous and evoked transmitter release was performedsimilarly as describedU-tests were used to compare two groups for alldata sets. Nonparametric Kruskal–Wallis tests were used for comparisonof more than two groups, followed by a Dunn's multiple comparisontest. P values, n values and U or K statistics aregiven in the figure legends or main text. Similarly, the electrophysiologicaldata are reported as mean±s.e.m. and P value denotes thesignificance according to one-way analysis of variance with Tukey'smultiple comparison post-test.Data were analysed using Prism (GraphPad Software). NonparametricMann–Whitney Author Information. The structure factors and atomic coordinates of theSpn-PDZ domain are deposited in the Protein Data Bank with accession number4XHV.How to cite this article: Muhammad, K. G. H. et al. Presynapticspinophilin tunes neurexin signalling to control active zone architecture andfunction. Nat. Commun. 6:8362 doi: 10.1038/ncomms9362 (2015).Supplementary Figures 1-12, Supplementary Table 1-2, Supplementary Methodsand Supplementary ReferencesLive movie of GCaMP5 fluorescence during 98 s of spontaneous activity at Control NMJsLive movie of GCaMP5 fluorescence during 35 episodes of AP stimulation at Control NMJsLive movie of GCaMP5 fluorescence during 98 s of spontaneous activity at Spn NMJsLive movie of GCaMP5 fluorescence during 35 episodes of AP stimulation at Spn NMJs