Involvement of autophagy in hypoxic-excitotoxic neuronal death.

Neuronal autophagy is increased in numerous excitotoxic conditions including neonatal cerebral hypoxia-ischemia (HI). However, the role of this HI-induced autophagy remains unclear. To clarify this role we established an in vitro model of excitotoxicity combining kainate treatment (Ka, 30 µM) with hypoxia (Hx, 6% oxygen) in primary neuron cultures. KaHx rapidly induced excitotoxic death that was completely prevented by MK801 or EGTA. KaHx also stimulated neuronal autophagic flux as shown by a rise in autophagosome number (increased levels of LC3-II and punctate LC3 labeling) accompanied by increases in lysosomal abundance and activity (increased SQSTM1/p62 degradation, and increased LC3-II levels in the presence of lysosomal inhibitors) and fusion (shown using an RFP-GFP-LC3 reporter). To determine the role of the enhanced autophagy we applied either pharmacological autophagy inhibitors (3-methyladenine or pepstatinA/E64) or lentiviral vectors delivering shRNAs targeting Becn1 or Atg7. Both strategies reduced KaHx-induced neuronal death. A prodeath role of autophagy was also confirmed by the enhanced toxicity of KaHx in cultures overexpressing BECN1 or ATG7. Finally, in vivo inhibition of autophagy by intrastriatal injection of a lentiviral vector expressing a Becn1-targeting shRNA increased the volume of intact striatum in a rat model of severe neonatal cerebral HI. These results clearly show a death-mediating role of autophagy in hypoxic-excitotoxic conditions and suggest that inhibition of autophagy should be considered as a neuroprotective strategy in HI brain injuries

Evaluation of the silver species nature in Ag-ITQ2 zeolites by the CO oxidation reaction

The authors thank the Spanish Ministry of Economy and Competitiveness through RTI2018-101784-B-I00 (MINECO/FEDER) and SEV-2016-0683 projects for the financial support. We gratefully acknowledge ALBA synchrotron for allocating beamtime (proposal 2015091414) and the CLAESS beamline staff for their help and technical support during our experiment. CG and NB thank the TUW Innovative Project GIP165CDGC. CG, SP, VT, NB and GR are thankful for financial support from the Austrian Science Fund (FWF) through projects DK+ Solids4Fun (W1243) and ComCat (I 1041-N28). I. Lopez Hernandez is grateful to Generalitat Valenciana and European Social Fund for the pre doctoral grant ACIF2017.López-Hernández, I.; García Yago, CI.; Truttmann, V.; Pollit, S.; Barrabés, N.; Rupprechter, G.; Rey Garcia, F.... (2020). Evaluation of the silver species nature in Ag-ITQ2 zeolites by the CO oxidation reaction. Catalysis Today. 345:22-26. https://doi.org/10.1016/j.cattod.2019.12.001S2226345Serhan, N., Tsolakis, A., Wahbi, A., Martos, F. J., & Golunski, S. (2019). Modifying catalytically the soot morphology and nanostructure in diesel exhaust: Influence of silver De-NOx catalyst (Ag/Al2O3). Applied Catalysis B: Environmental, 241, 471-482. doi:10.1016/j.apcatb.2018.09.068Góra-Marek, K., Tarach, K. A., Piwowarska, Z., Łaniecki, M., & Chmielarz, L. (2016). Ag-loaded zeolites Y and USY as catalysts for selective ammonia oxidation. Catalysis Science & Technology, 6(6), 1651-1660. doi:10.1039/c5cy01446hHu, X., Bai, J., Hong, H., & Li, C. (2016). Supercritical carbon dioxide anchored highly dispersed silver nanoparticles on 4A-zeolite and selective oxidation of styrene performance. CrystEngComm, 18(14), 2469-2476. doi:10.1039/c5ce02435hCerrillo, J. L., Palomares, A. E., Rey, F., Valencia, S., Pérez-Gago, M. B., Villamón, D., & Palou, L. (2018). Functional Ag-Exchanged Zeolites as Biocide Agents. ChemistrySelect, 3(17), 4676-4682. doi:10.1002/slct.201800432Dong, X.-Y., Gao, Z.-W., Yang, K.-F., Zhang, W.-Q., & Xu, L.-W. (2015). Nanosilver as a new generation of silver catalysts in organic transformations for efficient synthesis of fine chemicals. Catalysis Science & Technology, 5(5), 2554-2574. doi:10.1039/c5cy00285kSulaiman, K. O., Sudheeshkumar, V., & Scott, R. W. J. (2019). Activation of atomically precise silver clusters on carbon supports for styrene oxidation reactions. RSC Advances, 9(48), 28019-28027. doi:10.1039/c9ra05566eCoutiño-Gonzalez, E., Baekelant, W., Steele, J. A., Kim, C. W., Roeffaers, M. B. J., & Hofkens, J. (2017). Silver Clusters in Zeolites: From Self-Assembly to Ground-Breaking Luminescent Properties. Accounts of Chemical Research, 50(9), 2353-2361. doi:10.1021/acs.accounts.7b00295Liu, L., & Corma, A. (2018). Metal Catalysts for Heterogeneous Catalysis: From Single Atoms to Nanoclusters and Nanoparticles. Chemical Reviews, 118(10), 4981-5079. doi:10.1021/acs.chemrev.7b00776Zhao, J., & Jin, R. (2018). Heterogeneous catalysis by gold and gold-based bimetal nanoclusters. Nano Today, 18, 86-102. doi:10.1016/j.nantod.2017.12.009Zhang, B., Kaziz, S., Li, H., Hevia, M. G., Wodka, D., Mazet, C., … Barrabés, N. (2015). Modulation of Active Sites in Supported Au38(SC2H4Ph)24 Cluster Catalysts: Effect of Atmosphere and Support Material. The Journal of Physical Chemistry C, 119(20), 11193-11199. doi:10.1021/jp512022vZhang, B., Sels, A., Salassa, G., Pollitt, S., Truttmann, V., Rameshan, C., … Barrabés, N. (2018). Ligand Migration from Cluster to Support: A Crucial Factor for Catalysis by Thiolate‐protected Gold Clusters. ChemCatChem, 10(23), 5372-5376. doi:10.1002/cctc.201801474Natarajan, G., Mathew, A., Negishi, Y., Whetten, R. L., & Pradeep, T. (2015). A Unified Framework for Understanding the Structure and Modifications of Atomically Precise Monolayer Protected Gold Clusters. The Journal of Physical Chemistry C, 119(49), 27768-27785. doi:10.1021/acs.jpcc.5b08193Tsukuda, T., & Häkkinen, H. (2015). Introduction. Protected Metal Clusters - From Fundamentals to Applications, 1-7. doi:10.1016/b978-0-08-100086-1.00001-4Zhang, X., Qu, Z., Li, X., Wen, M., Quan, X., Ma, D., & Wu, J. (2010). Studies of silver species for low-temperature CO oxidation on Ag/SiO2 catalysts. Separation and Purification Technology, 72(3), 395-400. doi:10.1016/j.seppur.2010.03.012Kolobova, E., Pestryakov, A., Mamontov, G., Kotolevich, Y., Bogdanchikova, N., Farias, M., … Cortes Corberan, V. (2017). Low-temperature CO oxidation on Ag/ZSM-5 catalysts: Influence of Si/Al ratio and redox pretreatments on formation of silver active sites. Fuel, 188, 121-131. doi:10.1016/j.fuel.2016.10.037Ausavasukhi, A., Suwannaran, S., Limtrakul, J., & Sooknoi, T. (2008). Reversible interconversion behavior of Ag species in AgHZSM-5: XRD, 1H MAS NMR, TPR, TPHE, and catalytic studies. Applied Catalysis A: General, 345(1), 89-96. doi:10.1016/j.apcata.2008.04.026Shi, C., Cheng, M., Qu, Z., & Bao, X. (2005). On the correlation between microstructural changes of Ag-H-ZSM-5 catalysts and their catalytic performances in the selective catalytic reduction of NOx by methane. Journal of Molecular Catalysis A: Chemical, 235(1-2), 35-43. doi:10.1016/j.molcata.2004.10.045Afanasev, D. S., Yakovina, O. A., Kuznetsova, N. I., & Lisitsyn, A. S. (2012). High activity in CO oxidation of Ag nanoparticles supported on fumed silica. Catalysis Communications, 22, 43-47. doi:10.1016/j.catcom.2012.02.014Kolobova, E., Pestryakov, A., Shemeryankina, A., Kotolevich, Y., Martynyuk, O., Tiznado Vazquez, H. J., & Bogdanchikova, N. (2014). Formation of silver active states in Ag/ZSM-5 catalysts for CO oxidation. Fuel, 138, 65-71. doi:10.1016/j.fuel.2014.07.011Royer, S., & Duprez, D. (2010). Catalytic Oxidation of Carbon Monoxide over Transition Metal Oxides. ChemCatChem, 3(1), 24-65. doi:10.1002/cctc.201000378Soliman, N. K. (2019). Factors affecting CO oxidation reaction over nanosized materials: A review. Journal of Materials Research and Technology, 8(2), 2395-2407. doi:10.1016/j.jmrt.2018.12.012Du, M., Sun, D., Yang, H., Huang, J., Jing, X., Odoom-Wubah, T., … Li, Q. (2014). Influence of Au Particle Size on Au/TiO2 Catalysts for CO Oxidation. The Journal of Physical Chemistry C, 118(33), 19150-19157. doi:10.1021/jp504681fCorma, A., Fornés, V., Guil, J. ., Pergher, S., Maesen, T. L. ., & Buglass, J. . (2000). Preparation, characterisation and catalytic activity of ITQ-2, a delaminated zeolite. Microporous and Mesoporous Materials, 38(2-3), 301-309. doi:10.1016/s1387-1811(00)00149-9Joshi, C. P., Bootharaju, M. S., Alhilaly, M. J., & Bakr, O. M. (2015). [Ag25(SR)18]−: The «Golden» Silver Nanoparticle. Journal of the American Chemical Society, 137(36), 11578-11581. doi:10.1021/jacs.5b07088Aspromonte, S. G., Mizrahi, M. D., Schneeberger, F. A., López, J. M. R., & Boix, A. V. (2013). Study of the Nature and Location of Silver in Ag-Exchanged Mordenite Catalysts. Characterization by Spectroscopic Techniques. The Journal of Physical Chemistry C, 117(48), 25433-25442. doi:10.1021/jp4046269Veronesi, G., Deniaud, A., Gallon, T., Jouneau, P.-H., Villanova, J., Delangle, P., … Michaud-Soret, I. (2016). Visualization, quantification and coordination of Ag+ions released from silver nanoparticles in hepatocytes. Nanoscale, 8(38), 17012-17021. doi:10.1039/c6nr04381jVeronesi, G., Aude-Garcia, C., Kieffer, I., Gallon, T., Delangle, P., Herlin-Boime, N., … Carrière, M. (2015). Exposure-dependent Ag+release from silver nanoparticles and its complexation in AgS2sites in primary murine macrophages. Nanoscale, 7(16), 7323-7330. doi:10.1039/c5nr00353aHudson-Smith, N. V., Clement, P. L., Brown, R. P., Krause, M. O. P., Pedersen, J. A., & Haynes, C. L. (2016). Research highlights: speciation and transformations of silver released from Ag NPs in three species. Environmental Science: Nano, 3(6), 1236-1240. doi:10.1039/c6en90025aShimizu, K., Sugino, K., Kato, K., Yokota, S., Okumura, K., & Satsuma, A. (2007). Formation and Redispersion of Silver Clusters in Ag-MFI Zeolite as Investigated by Time-Resolved QXAFS and UV−Vis. The Journal of Physical Chemistry C, 111(4), 1683-1688. doi:10.1021/jp066995aChen, D., Qu, Z., Shen, S., Li, X., Shi, Y., Wang, Y., … Wu, J. (2011). Comparative studies of silver based catalysts supported on different supports for the oxidation of formaldehyde. Catalysis Today, 175(1), 338-345. doi:10.1016/j.cattod.2011.03.059Schuricht, F., & Reschetilowski, W. (2012). Simultaneous selective catalytic reduction (SCR) of NOx and N2O over Ag/ZSM-5 – Catalytic studies and mechanistic implications. Microporous and Mesoporous Materials, 164, 135-144. doi:10.1016/j.micromeso.2012.07.018Akolekar, D. B., & Bhargava, S. K. (2000). Adsorption of NO and CO on silver-exchanged microporous materials. Journal of Molecular Catalysis A: Chemical, 157(1-2), 199-206. doi:10.1016/s1381-1169(00)00055-8Liu, J., Krishna, K. S., Kumara, C., Chattopadhyay, S., Shibata, T., Dass, A., & Kumar, C. S. S. R. (2016). Understanding Au∼98Ag∼46(SR)60 nanoclusters through investigation of their electronic and local structure by X-ray absorption fine structure. RSC Advances, 6(30), 25368-25374. doi:10.1039/c5ra27396jChevrier, D. M., Yang, R., Chatt, A., & Zhang, P. (2015). Bonding properties of thiolate-protected gold nanoclusters and structural analogs from X-ray absorption spectroscopy. Nanotechnology Reviews, 4(2). doi:10.1515/ntrev-2015-0007Yamazoe, S., & Tsukuda, T. (2019). X-ray Absorption Spectroscopy on Atomically Precise Metal Clusters. Bulletin of the Chemical Society of Japan, 92(1), 193-204. doi:10.1246/bcsj.2018028

Ligand migration from cluster to support: a crucial factor for catalysis by Thiolate-protected gold clusters

Thiolate protected metal clusters are valuable precursors for the design of tailored nanosized catalysts. Their performance can be tuned precisely at atomic level, e.g. by the configuration/ type of ligands or by partial/complete removal of the ligand shell through controlled pre-treatment steps. However, the interaction between the ligand shell and the oxide support, as well as ligand removal by oxidative pre-treatment, are still poorly understood. Typically, it was assumed that the thiolate ligands are simply converted into SO 2 , CO 2 and H 2 O. Herein, we report the first detailed observation of sulfur ligand migration from Au to the oxide support upon deposition and oxidative pre-treatment, employing mainly S K-edge XANES. Conse- quently, thiolate ligand migration not only produces clean Au cluster surfaces but also the surrounding oxide support is modified by sulfur-containing species, with pronounced effects on catalytic propertiesPeer ReviewedPostprint (published version

BID-F1 and BID-F2 Domains of Bartonella henselae Effector Protein BepF Trigger Together with BepC the Formation of Invasome Structures

The gram-negative, zoonotic pathogen Bartonella henselae (Bhe) translocates seven distinct Bartonella effector proteins (Beps) via the VirB/VirD4 type IV secretion system (T4SS) into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation

Induced pseudoscalar coupling of the proton weak interaction

The induced pseudoscalar coupling $g_p$ is the least well known of the weak coupling constants of the proton's charged--current interaction. Its size is dictated by chiral symmetry arguments, and its measurement represents an important test of quantum chromodynamics at low energies. During the past decade a large body of new data relevant to the coupling $g_p$ has been accumulated. This data includes measurements of radiative and non radiative muon capture on targets ranging from hydrogen and few--nucleon systems to complex nuclei. Herein the authors review the theoretical underpinnings of $g_p$, the experimental studies of $g_p$, and the procedures and uncertainties in extracting the coupling from data. Current puzzles are highlighted and future opportunities are discussed.Comment: 58 pages, Latex, Revtex4, prepared for Reviews of Modern Physic

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Synthesis of Ethers via Reaction of Carbanions and Monoperoxyacetals

Although transfer of electrophilic alkoxyl (“RO+”) from organic peroxides to organometallics offers a complement to traditional methods for etherification, application has been limited by constraints associated with peroxide reactivity and stability. We now demonstrate that readily prepared tetrahydropyranyl monoperoxyacetals react with sp3 and sp2 organolithium and organomagnesium reagents to furnish moderate to high yields of ethers. The method is successfully applied to the synthesis of alkyl, alkenyl, aryl, heteroaryl, and cyclopropyl ethers, mixed O,O-acetals, and S,S,O-orthoesters. In contrast to reactions of dialkyl and alkyl/silyl peroxides, the displacements of monoperoxyacetals provide no evidence for alkoxy radical intermediates. At the same time, the high yields observed for transfer of primary, secondary, or tertiary alkoxides, the latter involving attack on neopentyl oxygen, are inconsistent with an SN2 mechanism. Theoretical studies suggest a mechanism involving Lewis acid promoted insertion of organometallics into the O−O bond

The dynamic structure of Au38(SR)24 nanoclusters supported on CeO2 upon pretreatment and CO oxidation

Atomically precise thiolate protected Au nanoclusters Au38(SC2H4Ph)24 on CeO2 were used for in-situ (operando) extended X-ray absorption fine structure/diffuse reflectance infrared fourier transform spectroscopy and ex situ scanning transmission electron microscopy–high-angle annular dark-field imaging/X-ray photoelectron spectroscopy studies monitoring cluster structure changes induced by activation (ligand removal) and CO oxidation. Oxidative pretreatment at 150 °C “collapsed” the clusters’ ligand shell, oxidizing the hydrocarbon backbone, but the S remaining on Au acted as poison. Oxidation at 250 °C produced bare Au surfaces by removing S which migrated to the support (forming Au+-S), leading to highest activity. During reaction, structural changes occurred via CO-induced Au and O-induced S migration to the support. The results reveal the dynamics of nanocluster catalysts and the underlying cluster chemistry.Peer ReviewedPostprint (author's final draft