Resonant Mie Scattering (RMieS) correction of infrared spectra from highly scattering biological samples

Infrared spectra of single biological cells often exhibit the “dispersion artefact” observed as a sharp decrease in intensity on the high wavenumber side of absorption bands, in particular the Amide I band at ~1655 cm-1, causing a downward shift of the true peak position. The presence of this effect makes any biochemical interpretation of the spectra unreliable. Recent theory has shed light on the origins of the ‘dispersion artefact’ which has been attributed to resonant Mie scattering (RMieS). In this paper a preliminary algorithm for correcting RMieS is presented and evaluated using simulated data. Results show that the ‘dispersion artefact’ appears to be removed, however, the correction is not perfect. An iterative approach was subsequently implemented whereby the reference spectrum is improved after each iteration, resulting in a more accurate correction. Consequently the corrected spectra become increasingly more representative of the pure absorbance spectra. Using this correction method reliable peak positions can be obtained

Direct and cytokine-mediated effects of albumin-fused growth hormone, TV-1106, on CYP enzyme expression in human hepatocytes in vitro

Some biologics can modulate cytokines that may lead to changes in expression of drugmetabolizing enzymes and cause drug-drug interactions (DDI). DDI potential of TV- 1106—an albumin-fused growth hormone (GH)—was investigated. In this study, human blood was exposed to recombinant human growth hormone (rhGH) or TV-1106, followed by isolation of the plasma and its application to human hepatocytes. While the treatment of blood with rhGH increased multiple cytokines, treatment of blood with TV-1106 had no effect on any of the nine cytokines tested. The interleukin (IL)-6 concentration was higher in the rhGH then in the TV-1106-treated plasma (P < .05). While rhGH had little or no effect on CYP1A2 or CYP2C19 mRNA but increased CYP3A4 mRNA twofold, TV-1106 had little or no effect on cytochrome P450 (CYP) mRNAs in hepatocytes. Although the plasma from rhGH-treated blood lowered CYP1A2 activity, the TV-1106 plasma had no effect on CYP activities. The CYP1A2 activity was lower in the rhGH- then in the TV-1106-plasma treated hepatocytes (P < .05). The results indicated that fusing GH with albumin made TV-1106 an unlikely participant of CYP1A2, CYP2C19 or CYP3A4-facilitated, direct or cytokine-driven DDI.The authors would like to thank scientists in the Scientific Operations at Sekisui XenoTech for their technical assistance

Selective synthesis of citrus flavonoids prunin and naringenin using heterogeneized biocatalyst on graphene oxide

[EN] Production of citrus flavonoids prunin and naringenin was performed selectively through the enzyme hydrolysis of naringin, a flavonoid glycoside abundant in grapefruit wastes. To produce the monoglycoside flavonoid, prunin, crude naringinase from Penicillium decumbens was purified by a single purification step resulting in an enzyme with high -rhamnosidase activity. Both crude and purified enzymes were covalently immobilized on graphene oxide. The activity of the immobilized enzymes at different pH levels and temperatures, and the thermal stability were determined and compared with those exhibited by the free naringinases using specific substrates: p-nitrophenyl--d-glucoside (Glc-pNP) and p-nitrophenyl-alpha-l-rhamnopyranoside (Rha-pNP). The crude and purified naringinase supported on GO were tested in the hydrolysis of naringin, giving naringenin and prunin, respectively, in excellent yields. The supported enzymes can be reused many times without loss of activity. The naringinase stabilized on GO has high potential to produce the valuable citrus flavonoids prunin and naringenin.Authors acknowledge the financial support from MICINN Project CTQ-2015-67592-P and Program Severo Ochoa (SEV-2016-0683). JVC thanks Universitat Politecnica de Valencia for predoctoral fellowships. JY and AC thank the support from the National Natural Science Foundation of China (Grant No. 21320102001) and the 111 Project (Grant No. B17020).Carceller-Carceller, JM.; Martínez Galán, JP.; Monti, R.; Bassan, JC.; Filice, M.; Iborra Chornet, S.; Yu, J.... (2019). Selective synthesis of citrus flavonoids prunin and naringenin using heterogeneized biocatalyst on graphene oxide. Green Chemistry. 21(4):839-849. https://doi.org/10.1039/c8gc03661fS839849214Puri, M., & Banerjee, U. C. (2000). Production, purification, and characterization of the debittering enzyme naringinase. Biotechnology Advances, 18(3), 207-217. doi:10.1016/s0734-9750(00)00034-3Vila-Real, H., Alfaia, A. J., Rosa, M. E., Calado, A. R., & Ribeiro, M. H. L. (2010). An innovative sol–gel naringinase bioencapsulation process for glycosides hydrolysis. Process Biochemistry, 45(6), 841-850. doi:10.1016/j.procbio.2010.02.004C. Grassin and P.Fauquembergue , in Industrial Enzymology , ed. S. West and T. Godfrey , Nature Publishing Group , New York , 2nd edn, 1996 , p. 225Tsen, H.-Y., & Tsai, S.-Y. (1988). Comparison of the kinetics and factors affecting the stabilities of chitin-immobilized naringinases from two fungal sources. Journal of Fermentation Technology, 66(2), 193-198. doi:10.1016/0385-6380(88)90047-7SOARES, N. F. F., & HOTCHKISS, J. H. (1998). Naringinase Immobilization in Packaging Films for Reducing Naringin Concentration in Grapefruit Juice. Journal of Food Science, 63(1), 61-65. doi:10.1111/j.1365-2621.1998.tb15676.xPuri, M., Kaur, H., & Kennedy, J. F. (2005). Covalent immobilization of naringinase for the transformation of a flavonoid. Journal of Chemical Technology & Biotechnology, 80(10), 1160-1165. doi:10.1002/jctb.1303Norouzian, D., Hosseinzadeh, A., Inanlou, D. N., & Moazami, N. (1999). World Journal of Microbiology and Biotechnology, 15(4), 501-502. doi:10.1023/a:1008980018481Nishita, M., Park, S.-Y., Nishio, T., Kamizaki, K., Wang, Z., Tamada, K., … Minami, Y. (2017). Ror2 signaling regulates Golgi structure and transport through IFT20 for tumor invasiveness. Scientific Reports, 7(1). doi:10.1038/s41598-016-0028-xZhang, Y., Wu, C., Guo, S., & Zhang, J. (2013). Interactions of graphene and graphene oxide with proteins and peptides. Nanotechnology Reviews, 2(1), 27-45. doi:10.1515/ntrev-2012-0078Mathesh, M., Luan, B., Akanbi, T. O., Weber, J. K., Liu, J., Barrow, C. J., … Yang, W. (2016). Opening Lids: Modulation of Lipase Immobilization by Graphene Oxides. ACS Catalysis, 6(7), 4760-4768. doi:10.1021/acscatal.6b00942Li, W., Wen, H., Shi, Q., & Zheng, G. (2016). Study on immobilization of (+) γ-lactamase using a new type of epoxy graphene oxide carrier. Process Biochemistry, 51(2), 270-276. doi:10.1016/j.procbio.2015.11.030Hong, S.-G., Kim, J. H., Kim, R. E., Kwon, S.-J., Kim, D. W., Jung, H.-T., … Kim, J. (2016). Immobilization of glucose oxidase on graphene oxide for highly sensitive biosensors. Biotechnology and Bioprocess Engineering, 21(4), 573-579. doi:10.1007/s12257-016-0373-4Liu, F., Piao, Y., Choi, K. S., & Seo, T. S. (2012). Fabrication of free-standing graphene composite films as electrochemical biosensors. Carbon, 50(1), 123-133. doi:10.1016/j.carbon.2011.07.061Wang, Z., Zhou, X., Zhang, J., Boey, F., & Zhang, H. (2009). Direct Electrochemical Reduction of Single-Layer Graphene Oxide and Subsequent Functionalization with Glucose Oxidase. The Journal of Physical Chemistry C, 113(32), 14071-14075. doi:10.1021/jp906348xSingh, R. K., Kumar, R., & Singh, D. P. (2016). Graphene oxide: strategies for synthesis, reduction and frontier applications. RSC Advances, 6(69), 64993-65011. doi:10.1039/c6ra07626bVila-Real, H., Alfaia, A. J., Bronze, M. R., Calado, A. R. T., & Ribeiro, M. H. L. (2011). Enzymatic Synthesis of the Flavone Glucosides, Prunin and Isoquercetin, and the Aglycones, Naringenin and Quercetin, with Selective α-L-Rhamnosidase and β-D-Glucosidase Activities of Naringinase. Enzyme Research, 2011, 1-11. doi:10.4061/2011/692618Mamma, D., Kalogeris, E., Hatzinikolaou, D. G., Lekanidou, A., Kekos, D., Macris, B. J., & Christakopoulos, P. (2004). Biochemical Characterization of the Multi-enzyme System Produced byPenicillium decumbensGrown on Rutin. Food Biotechnology, 18(1), 1-18. doi:10.1081/fbt-120030382Chang, H.-Y., Lee, Y.-B., Bae, H.-A., Huh, J.-Y., Nam, S.-H., Sohn, H.-S., … Lee, S.-B. (2011). Purification and characterisation of Aspergillus sojae naringinase: The production of prunin exhibiting markedly enhanced solubility with in vitro inhibition of HMG-CoA reductase. Food Chemistry, 124(1), 234-241. doi:10.1016/j.foodchem.2010.06.024Yadav, S., Yadava, S., & Yadav, K. D. S. (2013). Purification and characterization of α-l-rhamnosidase from Penicillium corylopholum MTCC-2011. Process Biochemistry, 48(9), 1348-1354. doi:10.1016/j.procbio.2013.05.001Zhu, Y., Jia, H., Xi, M., Xu, L., Wu, S., & Li, X. (2017). Purification and characterization of a naringinase from a newly isolated strain of Bacillus amyloliquefaciens 11568 suitable for the transformation of flavonoids. Food Chemistry, 214, 39-46. doi:10.1016/j.foodchem.2016.06.108Zhang, T., Yuan, W., Li, M., Miao, M., & Mu, W. (2018). Purification and characterization of an intracellular α-l-rhamnosidase from a newly isolated strain, Alternaria alternata SK37.001. Food Chemistry, 269, 63-69. doi:10.1016/j.foodchem.2018.06.134Vila-Real, H., Alfaia, A. J., Rosa, J. N., Gois, P. M. P., Rosa, M. E., Calado, A. R. T., & Ribeiro, M. H. (2011). α-Rhamnosidase and β-glucosidase expressed by naringinase immobilized on new ionic liquid sol–gel matrices: Activity and stability studies. Journal of Biotechnology, 152(4), 147-158. doi:10.1016/j.jbiotec.2010.08.005Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., … Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 150(1), 76-85. doi:10.1016/0003-2697(85)90442-7Erickson, H. P. (2009). Size and Shape of Protein Molecules at the Nanometer Level Determined by Sedimentation, Gel Filtration, and Electron Microscopy. Biological Procedures Online, 11(1), 32-51. doi:10.1007/s12575-009-9008-xZhang, J., Zhang, F., Yang, H., Huang, X., Liu, H., Zhang, J., & Guo, S. (2010). Graphene Oxide as a Matrix for Enzyme Immobilization. Langmuir, 26(9), 6083-6085. doi:10.1021/la904014zMarolewski, A. (1996). Fundamentals of Enzyme Kinetics. Revised Edition By Athel Cornish-Bowden. Portland Press, London. 1995. xiii + 343 pp. 17.5 cm × 24.5 cm. ISBN 1-85578-072-0. $29.00. Journal of Medicinal Chemistry, 39(4), 1010-1011. doi:10.1021/jm9508447Romero, C., Manjón, A., Bastida, J., & Iborra, J. (1985). A method for assaying the rhamnosidase activity of naringinase. Analytical Biochemistry, 149(2), 566-571. doi:10.1016/0003-2697(85)90614-1Fox, D. W., Savage, W. L., & Wender, S. H. (1953). Hydrolysis of Some Flavonoid Rhamnoglucosides to Flavonoid Glucosides. Journal of the American Chemical Society, 75(10), 2504-2505. doi:10.1021/ja01106a507Miller, G. L. (1959). Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry, 31(3), 426-428. doi:10.1021/ac60147a030LAEMMLI, U. K. (1970). Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 227(5259), 680-685. doi:10.1038/227680a0Heukeshoven, J., & Dernick, R. (1985). Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis, 6(3), 103-112. doi:10.1002/elps.1150060302Sheldon, R. A., & van Pelt, S. (2013). Enzyme immobilisation in biocatalysis: why, what and how. Chem. Soc. Rev., 42(15), 6223-6235. doi:10.1039/c3cs60075

Covalent Immobilization of Naringinase over Two-Dimensional 2D Zeolites and its Applications in a Continuous Process to Produce Citrus Flavonoids and for Debittering of Juices

This is the peer reviewed version of the following article: J. M. Carceller, J. P. Martínez Galán, R. Monti, J. C. Bassan, M. Filice, J. Yu, M. J. Climent, S. Iborra, A. Corma, ChemCatChem 2020, 12, 4502, which has been published in final form at https://doi.org/10.1002/cctc.202000320. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] The crude naringinase from Penicillium decumbens and a purified naringinase with high a-L-rhamnosidase activity could be covalently immobilized on two-dimensional zeolite ITQ-2 after surface modification with glutaraldehyde. The influence of pH and temp. on the enzyme activity (in free and immobilized forms) as well as the thermal stability were detd. using the specific substrate: p-nitrophenyl-alpha-L-rhamnopyranoside (Rha-pNP). The crude and purified naringinase supported on ITQ-2 were applied in the hydrolysis of naringin, giving the flavonoids naringenin and prunin resp. with a conversion >90% and excellent selectivity. The supported enzymes showed long term stability, being possible to perform up to 25 consecutive cycles without loss of activity, showing its high potential to produce the valuable citrus flavonoids prunin and naringenin. We have also succeeded in the application of the immobilized crude naringinase on ITQ-2 for debittering grapefruit juices in a continuous process that was maintained operating for 300 h, with excellent results.The authors acknowledge financial support from PGC2018-097277-B-100 (MCIU/AEI/FEDER,UE) project and Program Severo Ochoa (SEV-2016-0683). Jilin agreement 111 Project (Grant No. B17020). JMC thanks to Universitat Politecnica de Valencia for predoctoral fellowships.Carceller-Carceller, JM.; Martínez Galán, JP.; Monti, R.; Bassan, JC.; Filice, M.; Yu, J.; Climent Olmedo, MJ.... (2020). Covalent Immobilization of Naringinase over Two-Dimensional 2D Zeolites and its Applications in a Continuous Process to Produce Citrus Flavonoids and for Debittering of Juices. ChemCatChem. 12(18):4502-4511. https://doi.org/10.1002/cctc.202000320S450245111218Puri, M., & Banerjee, U. C. (2000). Production, purification, and characterization of the debittering enzyme naringinase. Biotechnology Advances, 18(3), 207-217. doi:10.1016/s0734-9750(00)00034-3Vila-Real, H., Alfaia, A. J., Rosa, M. E., Calado, A. R., & Ribeiro, M. H. L. (2010). An innovative sol–gel naringinase bioencapsulation process for glycosides hydrolysis. Process Biochemistry, 45(6), 841-850. doi:10.1016/j.procbio.2010.02.004RoitNer, M., Schalkhammer, T., & Pittner, F. (1984). Preparation of prunin with the help of immobilized naringinase pretreated with alkaline buffer. Applied Biochemistry and Biotechnology, 9(5-6), 483-488. doi:10.1007/bf02798402Ribeiro, I. A., Rocha, J., Sepodes, B., Mota-Filipe, H., & Ribeiro, M. H. (2008). Effect of naringin enzymatic hydrolysis towards naringenin on the anti-inflammatory activity of both compounds. Journal of Molecular Catalysis B: Enzymatic, 52-53, 13-18. doi:10.1016/j.molcatb.2007.10.011Puri, M., Marwaha, S. S., Kothari, R. M., & Kennedy, J. F. (1996). Biochemical Basis of Bitterness in Citrus Fruit Juices and Biotech Approaches for Debittering. Critical Reviews in Biotechnology, 16(2), 145-155. doi:10.3109/07388559609147419Barbosa, O., Ortiz, C., Berenguer-Murcia, Á., Torres, R., Rodrigues, R. C., & Fernandez-Lafuente, R. (2015). Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts. Biotechnology Advances, 33(5), 435-456. doi:10.1016/j.biotechadv.2015.03.006Garcia-Galan, C., Berenguer-Murcia, Á., Fernandez-Lafuente, R., & Rodrigues, R. C. (2011). Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance. Advanced Synthesis & Catalysis, 353(16), 2885-2904. doi:10.1002/adsc.201100534ONO, M., TOSA, T., & CHIBATA, I. (1978). Preparation and properties of immobilized naringinase using tannin-aminohexyl cellulose. Agricultural and Biological Chemistry, 42(10), 1847-1853. doi:10.1271/bbb1961.42.1847Tsen, H.-Y., & Tsai, S.-Y. (1988). Comparison of the kinetics and factors affecting the stabilities of chitin-immobilized naringinases from two fungal sources. Journal of Fermentation Technology, 66(2), 193-198. doi:10.1016/0385-6380(88)90047-7SOARES, N. F. F., & HOTCHKISS, J. H. (1998). Naringinase Immobilization in Packaging Films for Reducing Naringin Concentration in Grapefruit Juice. Journal of Food Science, 63(1), 61-65. doi:10.1111/j.1365-2621.1998.tb15676.xPuri, M., Kaur, H., & Kennedy, J. F. (2005). Covalent immobilization of naringinase for the transformation of a flavonoid. Journal of Chemical Technology & Biotechnology, 80(10), 1160-1165. doi:10.1002/jctb.1303Lei, S., Xu, Y., Fan, G., Xiao, M., & Pan, S. (2011). Immobilization of naringinase on mesoporous molecular sieve MCM-41 and its application to debittering of white grapefruit. Applied Surface Science, 257(9), 4096-4099. doi:10.1016/j.apsusc.2010.12.003Luo, J., Li, Q., Sun, X., Tian, J., Fei, X., Shi, F., … Liu, X. (2019). The study of the characteristics and hydrolysis properties of naringinase immobilized by porous silica material. RSC Advances, 9(8), 4514-4520. doi:10.1039/c9ra00075eNunes, M. A. P., Vila-Real, H., Fernandes, P. C. B., & Ribeiro, M. H. L. (2009). Immobilization of Naringinase in PVA–Alginate Matrix Using an Innovative Technique. Applied Biochemistry and Biotechnology, 160(7), 2129-2147. doi:10.1007/s12010-009-8733-6Busto, M. D., Meza, V., Ortega, N., & Perez-Mateos, M. (2007). Immobilization of naringinase from Aspergillus niger CECT 2088 in poly(vinyl alcohol) cryogels for the debittering of juices. Food Chemistry, 104(3), 1177-1182. doi:10.1016/j.foodchem.2007.01.033Huang, W., Zhan, Y., Shi, X., Chen, J., Deng, H., & Du, Y. (2017). Controllable immobilization of naringinase on electrospun cellulose acetate nanofibers and their application to juice debittering. International Journal of Biological Macromolecules, 98, 630-636. doi:10.1016/j.ijbiomac.2017.02.018Gong, X., Xie, W., Wang, B., Gu, L., Wang, F., Ren, X., … Yang, L. (2017). Altered spontaneous calcium signaling of in situ chondrocytes in human osteoarthritic cartilage. Scientific Reports, 7(1). doi:10.1038/s41598-017-17172-wCarceller, J. M., Martínez Galán, J. P., Monti, R., Bassan, J. C., Filice, M., Iborra, S., … Corma, A. (2019). Selective synthesis of citrus flavonoids prunin and naringenin using heterogeneized biocatalyst on graphene oxide. Green Chemistry, 21(4), 839-849. doi:10.1039/c8gc03661fPuri, M., Marwaha, S. S., & Kothari, R. M. (1996). Studies on the applicability of alginate-entrapped naringiase for the debittering of kinnow juice. Enzyme and Microbial Technology, 18(4), 281-285. doi:10.1016/0141-0229(95)00100-xNorouzian, D., Hosseinzadeh, A., Inanlou, D. N., & Moazami, N. (1999). World Journal of Microbiology and Biotechnology, 15(4), 501-502. doi:10.1023/a:1008980018481Saallah, S., Naim, M. N., Lenggoro, I. W., Mokhtar, M. N., Abu Bakar, N. F., & Gen, M. (2016). Immobilisation of cyclodextrin glucanotransferase into polyvinyl alcohol (PVA) nanofibres via electrospinning. Biotechnology Reports, 10, 44-48. doi:10.1016/j.btre.2016.03.003Cipolatti, E. P., Valério, A., Henriques, R. O., Moritz, D. E., Ninow, J. L., Freire, D. M. G., … de Oliveira, D. (2016). Nanomaterials for biocatalyst immobilization – state of the art and future trends. RSC Advances, 6(106), 104675-104692. doi:10.1039/c6ra22047aCorma, A., Fornes, V., & Rey, F. (2002). Delaminated Zeolites: An Efficient Support for Enzymes. Advanced Materials, 14(1), 71-74. doi:10.1002/1521-4095(20020104)14:13.0.co;2-wGallego, E. M., Portilla, M. T., Paris, C., León-Escamilla, A., Boronat, M., Moliner, M., & Corma, A. (2017). «Ab initio» synthesis of zeolites for preestablished catalytic reactions. Science, 355(6329), 1051-1054. doi:10.1126/science.aal0121Margarit, V. J., Díaz-Rey, M. R., Navarro, M. T., Martínez, C., & Corma, A. (2018). Direct Synthesis of Nano-Ferrierite along the 10-Ring-Channel Direction Boosts Their Catalytic Behavior. Angewandte Chemie, 130(13), 3517-3521. doi:10.1002/ange.201711418Barbosa, O., Ortiz, C., Berenguer-Murcia, Á., Torres, R., Rodrigues, R. C., & Fernandez-Lafuente, R. (2014). Glutaraldehyde in bio-catalysts design: a useful crosslinker and a versatile tool in enzyme immobilization. RSC Adv., 4(4), 1583-1600. doi:10.1039/c3ra45991hSmith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., … Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 150(1), 76-85. doi:10.1016/0003-2697(85)90442-7Marolewski, A. (1996). Fundamentals of Enzyme Kinetics. Revised Edition By Athel Cornish-Bowden. Portland Press, London. 1995. xiii + 343 pp. 17.5 cm × 24.5 cm. ISBN 1-85578-072-0. $29.00. Journal of Medicinal Chemistry, 39(4), 1010-1011. doi:10.1021/jm9508447Miller, G. L. (1959). Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry, 31(3), 426-428. doi:10.1021/ac60147a030Cheong, M. W., Liu, S. Q., Zhou, W., Curran, P., & Yu, B. (2012). Chemical composition and sensory profile of pomelo (Citrus grandis (L.) Osbeck) juice. Food Chemistry, 135(4), 2505-2513. doi:10.1016/j.foodchem.2012.07.012Fox, D. W., Savage, W. L., & Wender, S. H. (1953). Hydrolysis of Some Flavonoid Rhamnoglucosides to Flavonoid Glucosides. Journal of the American Chemical Society, 75(10), 2504-2505. doi:10.1021/ja01106a507Corma, A., Fornes, V., Pergher, S. B., Maesen, T. L. M., & Buglass, J. G. (1998). Delaminated zeolite precursors as selective acidic catalysts. Nature, 396(6709), 353-356. doi:10.1038/24592Camblor, M. A., Corma, A., & Valencia, S. (1998). Characterization of nanocrystalline zeolite Beta. Microporous and Mesoporous Materials, 25(1-3), 59-74. doi:10.1016/s1387-1811(98)00172-3Beck, J. S., Vartuli, J. C., Roth, W. J., Leonowicz, M. E., Kresge, C. T., Schmitt, K. D., … Schlenker, J. L. (1992). A new family of mesoporous molecular sieves prepared with liquid crystal templates. Journal of the American Chemical Society, 114(27), 10834-10843. doi:10.1021/ja00053a02

Interaction between the glutamate transporter GLT1b and the synaptic PDZ domain protein PICK1

This is the published version. Copyright WileySynaptic plasticity is implemented by the interaction of glutamate receptors with PDZ domain proteins. Glutamate transporters provide the only known mechanism of clearance of glutamate from excitatory synapses, and GLT1 is the major glutamate transporter. We show here that GLT1 interacts with the PDZ domain protein PICK1, which plays a critical role in regulating the expression of glutamate receptors at excitatory synapses. A yeast two-hybrid screen of a neuronal library using the carboxyl tail of GLT1b yielded clones expressing PICK1. The GLT1b C-terminal peptide bound to PICK1 with high affinity (Ki = 6.5 ± 0.4 μm) in an in vitro fluorescence polarization assay. We also tested peptides based on other variants of GLT1 and other glutamate transporters. GLT1b co-immunoprecipitated with PICK1 from rat brain lysates and COS7 cell lysates derived from cells transfected with plasmids expressing PICK1 and GLT1b. In addition, expression of GLT1b in COS7 cells changed the distribution of PICK1, bringing it to the surface. GLT1b and PICK1 co-localized with each other and with synaptic markers in hippocampal neurons in culture. Phorbol ester, an activator of protein kinase C (PKC), a known PICK1 interactor, had no effect on glutamate transport in rat forebrain neurons in culture. However, we found that exposure of neurons to a myristolated decoy peptide with sequence identical to the C-terminal sequence of GLT1b designed to block the PICK1–GLT1b interaction rendered glutamate transport into neurons responsive to phorbol ester. These results suggest that the PICK1–GLT1b interaction regulates the modulation of GLT1 function by PKC.The authors are grateful to Sara Vasquez who provided excellent technical assistance in preparing the neuronal cultures. In addition, we are grateful for helpful discussions with Drs Gabriel Corfas, Michael Berne and Michael Robinson, to Dr Tom Schwarz for reading an early version of this manuscript, and to Dr Jeff Rothstein for providing an anti-cGLT1a antibody. We are also indebted to Dr Robinson for providing us with a detailed protocol for the biotinylation studies. This work was funded by grants from the Ron Shapiro Charitable Foundation (P.A.R.), the Muscular Dystrophy Association (P.A.R.), and National Institutes of Health research grant NS 40753 and a Mental Retardation Core Grant HD18655

Effect of aliskiren on post-discharge outcomes among diabetic and non-diabetic patients hospitalized for heart failure: insights from the ASTRONAUT trial

Aims The objective of the Aliskiren Trial on Acute Heart Failure Outcomes (ASTRONAUT) was to determine whether aliskiren, a direct renin inhibitor, would improve post-discharge outcomes in patients with hospitalization for heart failure (HHF) with reduced ejection fraction. Pre-specified subgroup analyses suggested potential heterogeneity in post-discharge outcomes with aliskiren in patients with and without baseline diabetes mellitus (DM). Methods and results ASTRONAUT included 953 patients without DM (aliskiren 489; placebo 464) and 662 patients with DM (aliskiren 319; placebo 343) (as reported by study investigators). Study endpoints included the first occurrence of cardiovascular death or HHF within 6 and 12 months, all-cause death within 6 and 12 months, and change from baseline in N-terminal pro-B-type natriuretic peptide (NT-proBNP) at 1, 6, and 12 months. Data regarding risk of hyperkalaemia, renal impairment, and hypotension, and changes in additional serum biomarkers were collected. The effect of aliskiren on cardiovascular death or HHF within 6 months (primary endpoint) did not significantly differ by baseline DM status (P = 0.08 for interaction), but reached statistical significance at 12 months (non-DM: HR: 0.80, 95% CI: 0.64-0.99; DM: HR: 1.16, 95% CI: 0.91-1.47; P = 0.03 for interaction). Risk of 12-month all-cause death with aliskiren significantly differed by the presence of baseline DM (non-DM: HR: 0.69, 95% CI: 0.50-0.94; DM: HR: 1.64, 95% CI: 1.15-2.33; P < 0.01 for interaction). Among non-diabetics, aliskiren significantly reduced NT-proBNP through 6 months and plasma troponin I and aldosterone through 12 months, as compared to placebo. Among diabetic patients, aliskiren reduced plasma troponin I and aldosterone relative to placebo through 1 month only. There was a trend towards differing risk of post-baseline potassium ≥6 mmol/L with aliskiren by underlying DM status (non-DM: HR: 1.17, 95% CI: 0.71-1.93; DM: HR: 2.39, 95% CI: 1.30-4.42; P = 0.07 for interaction). Conclusion This pre-specified subgroup analysis from the ASTRONAUT trial generates the hypothesis that the addition of aliskiren to standard HHF therapy in non-diabetic patients is generally well-tolerated and improves post-discharge outcomes and biomarker profiles. In contrast, diabetic patients receiving aliskiren appear to have worse post-discharge outcomes. Future prospective investigations are needed to confirm potential benefits of renin inhibition in a large cohort of HHF patients without D