@article{17885, abstract = {The formation of new ribosomes is tightly coordinated with cell growth and proliferation. In eukaryotes, the correct assembly of all ribosomal proteins and RNAs follows an intricate scheme of maturation and rearrangement steps across three cellular compartments: the nucleolus, nucleoplasm, and cytoplasm. We demonstrate that usnic acid, a lichen secondary metabolite, inhibits the maturation of the large ribosomal subunit in yeast. We combine biochemical characterization of pre-ribosomal particles with a quantitative single-particle cryo-EM approach to monitor changes in nucleolar particle populations upon drug treatment. Usnic acid rapidly blocks the transition from nucleolar state B to C of Nsa1-associated pre-ribosomes, depleting key maturation factors such as Dbp10 and hindering pre-rRNA processing. This primary nucleolar block rapidly rebounds on earlier stages of the pathway which highlights the regulatory linkages between different steps. In summary, we provide an in-depth characterization of the effect of usnic acid on ribosome biogenesis, which may have implications for its reported anti-cancer activities.}, author = {Kofler, Lisa and Grundmann, Lorenz and Gerhalter, Magdalena and Prattes, Michael and Merl-Pham, Juliane and Zisser, Gertrude and Grishkovskaya, Irina and Hodirnau, Victor-Valentin and Vareka, Martin and Breinbauer, Rolf and Hauck, Stefanie M. and Haselbach, David and Bergler, Helmut}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{The novel ribosome biogenesis inhibitor usnic acid blocks nucleolar pre-60S maturation}}, doi = {10.1038/s41467-024-51754-3}, volume = {15}, year = {2024}, } @article{15084, abstract = {GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca 2+ -dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the “Flash and Freeze-fracture” method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals.}, author = {Koppensteiner, Peter and Bhandari, Pradeep and Önal, Hüseyin C and Borges Merjane, Carolina and Le Monnier, Elodie and Roy, Utsa and Nakamura, Yukihiro and Sadakata, Tetsushi and Sanbo, Makoto and Hirabayashi, Masumi and Rhee, JeongSeop and Brose, Nils and Jonas, Peter M and Shigemoto, Ryuichi}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, number = {8}, publisher = {National Academy of Sciences}, title = {{GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles}}, doi = {10.1073/pnas.2301449121}, volume = {121}, year = {2024}, } @phdthesis{18101, abstract = {The Retroviridae family consists of two sub-families, the Orthoretrovirinae and the Spumaretrovirinae. The Orthoretroviruses contain important human pathogens, such as the human immunodeficiency virus 1 (HIV-1). They also harbor other retrovirus species which are regularly used as model systems to study the retroviral life cycle. The main structural component of the retroviruses, is the Gag protein and its truncation derivatives occurring during viral maturation. Orthoretroviral Gag assemblies have been extensively studied to understand the interactions that confer stability and morphology to viral particles. The Spumaretrovirinae subfamily represent an early diverging branch of the Retroviridae. Its members, the Foamy viruses (FV), share most of the conventional features found in retroviruses. However, they also possess multiple characteristics that make them unique. In particular, FV Gag does not get extensively cleaved as in orthoretroviruses. Hence, the Gag architecture deviates from the canonical domain arrangement in FV. They also exhibit a peculiar particle morphology, having no apparent immature state and a seemingly icosahedral mature particle. Due to this, many fundamental questions on FV structural assembly mechanisms remain open. To answer these questions, was the main focus of this thesis. Mainly, it is not known how FV assemble their core in a virus particle and what are the important assembly interaction sites within said core. What is the minimum assembly competent domain of FV Gag? Is there a morphological change in the assembly type of FVGag lattices? If so, what is defining these morphological shifts? Finally, it would be interesting to know what is the evolutionary relationship between FV and the rest of the retrotranscribing elements, from a structural point of view? To answer these questions, membrane-enveloped mammalian cell-derived FV virus-like particles (VLPs) were produced. Cryo-electron tomography (cryo-ET) analysis suggested these FV VLPs do not form a canonical retroviral Gag lattice structure, which is in line with earlier observations. To further evaluate FV Gag assembly competence and morphology, the first bacterial cell-derived in vitro VLP assembly system was designed and optimized. Using this system with different truncation variants, the minimum assembly competent domain of FV Gag was found to be the putative CA300-477 domain. Varying VLP morphologies were also observed and strongly suggested residues upstream of CA300-477 play a role in morphology determination. Finally, a combined cryo-electron microscopy (cryoEM) and cryo-ET approach was taken to analyze tubular assemblies from the minimal assembly competent domain. This revealed an unexpectedly unique non-canonical assembly architecture. Three novel lattice stabilizing interfaces were described which proved to be as unique as the lattice arrangement. Comparison to a newly published FV CA core structure revealed the CA-CA interactions in the atypical assembly do not recapitulate what is described for the FV core lattice. However, the new in vitro VLP assembly system obtained in this thesis also provides an exciting opportunity to study still unresolved FV assembly features in a potentially facilitated approach compared to conventional methods. In summary, this work provided a deeper understanding of the basic FV Gag assembly unit, as well as presenting the first FV Gag-derived in vitro VLP assembly system. This system reveals a novel and unique assembly architecture among retroviral in vitro assemblies.}, author = {Porley, Dario J}, isbn = {978-3-99078-041-1}, issn = {2663-337X}, pages = {131}, publisher = {Institute of Science and Technology Austria}, title = {{Structural characterization of spumavirus capsid assemblies}}, doi = {10.15479/at:ista:18101}, year = {2024}, } @phdthesis{18477, abstract = {ADAR1 is broadly expressed across various tissues and is vital in regulating pathways associated with innate immune responses. ADAR1 marks double-stranded RNA as "self" through its A-to-I editing activity, effectively repressing autoimmunity and maintaining immune tolerance. This editing process has been detected at millions of sites across the human genome. However, the mechanism underlying ADAR1's substrate selectivity properties remains largely unclear, with much of the current knowledge derived from comparisons to its more extensively studied homolog, ADAR2. By studying ADAR1 in complex with its RNA substrates and applying a combination of biochemical techniques and structural studies using CryoEM, we aim to gain a more comprehensive understanding of the substrate selectivity characteristics of ADAR1. In this thesis, the purification protocol for ADAR1 was successfully optimized, resulting in the first report in the literature to achieve high protein purity and activity. This advancement enabled the investigation of complex formation between ADAR1 and various RNA substrates, leading to the identification of optimal conditions for preparing the cryoEM sample. However, despite comprehensive optimization of the cryo-EM conditions, the resulting data lacked the desired quality, highlighting the need for similar rigorous optimization of the RNA substrates to facilitate structural studies of the ADAR1-RNA complex. The study was complemented by AlphaFold predictions, which provided some insights into this mechanism. Moreover, during this project I established a collaboration with a research group focused on studying ADAR homologs. Notably ADAR homologs were identified in bivalve species, and it was further demonstrated that ADAR and its A-to-I editing activity are upregulated in Pacific oysters during infections with Ostreid herpesvirus-1—a highly infectious virus that leads to significant losses in oyster populations globally. I successfully purified oyster ADAR and prepared in vitro edited RNA for nanopore sequencing—a direct sequencing technology capable of detecting modified nucleotides without the need for reverse transcription. The collaborators initiated optimization of this nanopore-based approach. However, current technological limitations still constrain the reliable detection of modified nucleotides. The project also examined the impact of RNA editing on RNA binding and filament formation by MDA5, a key cytosolic dsRNA sensor that triggers an interferon response. A primary target of ADAR1's editing activity is RNA derived from repetitive elements present in the genome, particularly Alu elements forming double-stranded RNA. When unedited, these RNA sequences are recognized by MDA5. However, the mechanisms by which MDA5 interacts with Alu RNAs, as well as the role of A-to-I editing in influencing this binding, are still not well understood. The interaction between MDA5 and Alu elements, was successfully established. This was achieved through the testing of different RNA variants and the evaluation of filament formation using binding techniques and electron microscopy imaging. This groundwork has set the conditions for further evaluation using CryoEM. Furthermore, the effects of A-to-I editing on the binding properties of MDA5 with Alu RNA were investigated. Given the recent research that has provided new insights into MDA5's interaction with dsRNA, it is essential to revise the experimental setup to integrate these findings before moving forward with the CryoEM sample analysis.}, author = {Kaczmarek, Beata M}, isbn = {978-3-99078-045-9}, issn = {2663-337X}, pages = {124}, publisher = {Institute of Science and Technology Austria}, title = {{Biochemical and structural insights into ADAR1 RNA editing}}, doi = {10.15479/at:ista:18477}, year = {2024}, } @article{14843, abstract = {The coupling between Ca2+ channels and release sensors is a key factor defining the signaling properties of a synapse. However, the coupling nanotopography at many synapses remains unknown, and it is unclear how it changes during development. To address these questions, we examined coupling at the cerebellar inhibitory basket cell (BC)-Purkinje cell (PC) synapse. Biophysical analysis of transmission by paired recording and intracellular pipette perfusion revealed that the effects of exogenous Ca2+ chelators decreased during development, despite constant reliance of release on P/Q-type Ca2+ channels. Structural analysis by freeze-fracture replica labeling (FRL) and transmission electron microscopy (EM) indicated that presynaptic P/Q-type Ca2+ channels formed nanoclusters throughout development, whereas docked vesicles were only clustered at later developmental stages. Modeling suggested a developmental transformation from a more random to a more clustered coupling nanotopography. Thus, presynaptic signaling developmentally approaches a point-to-point configuration, optimizing speed, reliability, and energy efficiency of synaptic transmission.}, author = {Chen, JingJing and Kaufmann, Walter and Chen, Chong and Arai, Itaru and Kim, Olena and Shigemoto, Ryuichi and Jonas, Peter M}, issn = {1097-4199}, journal = {Neuron}, number = {5}, pages = {755--771.e9}, publisher = {Elsevier}, title = {{Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse}}, doi = {10.1016/j.neuron.2023.12.002}, volume = {112}, year = {2024}, } @phdthesis{15101, abstract = {The coupling between presynaptic Ca2+ channels and release sensors is a key factor that determines speed and efficacy of synapse transmission. At some excitatory synapses, channel–sensor coupling becomes tighter during development, and tightening is often associated with a switch in the reliance on different Ca2+ channel subtypes. However, the coupling topography at many synapses remains unknown, and it is unclear how it changes during development. To address this question, we analyzed the coupling configuration at the cerebellar basket cell (BC) to Purkinje cell (PC) synapse at different developmental stages, combining biophysical analysis, structural analysis, and modeling. Quantal analysis of BC–PC indicated that release probability decreased, while the number of functional sites increased during development. Although transmitter release persistently relied on P/Q-type Ca2+ channels in the time period postnatal day 7–23, effects of the Ca2+ chelator EGTA and BAPTA applied by intracellular pipette perfusion decreased during development, indicative of tightening of source-sensor coupling. Furthermore, presynaptic action potentials became shorter during development, suggesting reduced efficacy of Ca2+ channel activation. Structural analysis by freeze-fracture replica labeling (FRL) and transmission electron microscopy (EM) indicated that presynaptic P/Q-type Ca2+ channels formed nanoclusters throughout development, whereas docked vesicles were only clustered at later developmental stages. The number of functional release sites correlated better with the AZ number early in development, but match better with the Ca2+ channel cluster number at later stages. Modeling suggested a developmental transformation from a more random to a more clustered coupling nanotopography. Thus, presynaptic signaling developmentally approaches a point-to-point configuration, optimizing speed, reliability, and energy efficiency of synaptic transmission.}, author = {Chen, JingJing}, issn = {2663-337X}, pages = {84}, publisher = {Institute of Science and Technology Austria}, title = {{Developmental transformation of nanodomain coupling between Ca2+ channels and release sensors at a central GABAergic synapse}}, doi = {10.15479/at:ista:15101}, year = {2024}, } @article{15323, abstract = {Supercomplexes of the respiratory chain are established constituents of the oxidative phosphorylation system, but their role in mammalian metabolism has been hotly debated. Although recent studies have shown that different tissues/organs are equipped with specific sets of supercomplexes, depending on their metabolic needs, the notion that supercomplexes have a role in the regulation of metabolism has been challenged. However, irrespective of the mechanistic conclusions, the composition of various high molecular weight supercomplexes remains uncertain. Here, using cryogenic electron microscopy, we demonstrate that mammalian (mouse) tissues contain three defined types of ‘respirasome’, supercomplexes made of CI, CIII2 and CIV. The stoichiometry and position of CIV differs in the three respirasomes, of which only one contains the supercomplex-associated factor SCAF1, whose involvement in respirasome formation has long been contended. Our structures confirm that the ‘canonical’ respirasome (the C-respirasome, CICIII2CIV) does not contain SCAF1, which is instead associated to a different respirasome (the CS-respirasome), containing a second copy of CIV. We also identify an alternative respirasome (A-respirasome), with CIV bound to the ‘back’ of CI, instead of the ‘toe’. This structural characterization of mouse mitochondrial supercomplexes allows us to hypothesize a mechanistic basis for their specific role in different metabolic conditions.}, author = {Vercellino, Irene and Sazanov, Leonid A}, issn = {1545-9985}, journal = {Nature Structural and Molecular Biology}, pages = {1061--1071}, publisher = {Springer Nature}, title = {{SCAF1 drives the compositional diversity of mammalian respirasomes}}, doi = {10.1038/s41594-024-01255-0}, volume = {31}, year = {2024}, } @phdthesis{17319, abstract = {This thesis comprises two distinct projects, each offering unique insights into fundamental cellular processes. While distinct in their focus, these different perspectives have a common theme: chemiosmotic theory and utilisation of the proton gradient for driving the essential processes like auxin efflux and ATP synthesis, effectively bridging the membrane protein structure and function from the realms of plant biology and cellular bioenergetics. The first project of this thesis centres on the characterisation of PIN proteins, a class of transmembrane transporters pivotal in the regulation of auxin transport and distribution in plants. PINs form a conserved and phylogenetically abundant group of transporters present in land plants and certain algae. Despite their great importance, they were one of the few elusive proteins essential for plant development not to be structurally and mechanistically characterised since their discovery almost 30 years ago. This work aimed to uncover the structural and functional dynamics of the PIN protein-mediated auxin transport using an array of experimental techniques, including protein purification, biochemical assays and structural analysis. Through an exhaustive screening process that took several years and included testing different PIN homologues, expression systems, constructs, and purification conditions, we developed a robust protocol for isolating the pure, stable, and monodisperse PIN8 protein. Moreover, utilising biophysical methods and buffer screening, we demonstrated that PIN8 exhibits detergent and pH-dependent stability, with mild detergents and lower pH (5.0 and 6.0) being optimal for the stability of the protein. Using SEC-MALS and crosslinking, we determined that PIN8 forms dimers, which was confirmed by our structural studies. We obtained a cryo-EM map of PIN8 at pH 6.0, and, compared to recently published structures, our map implies major pH-dependent conformational changes and possibly utilisation of the proton gradient in the transport mechanism. The subject of the second project was F1Fo-ATP synthase, an enzyme complex fundamental to cellular energy metabolism. Through an approach integrating biochemical assays and structural analysis, this research aimed to unveil the molecular mechanism of inhibition of ATP synthase by yaku´amide, a bioactive compound with potential therapeutic implications. Using submitochondrial particles and purified F1Fo-ATP synthase, we demonstrated that, contrary to published data, yaku´amide inhibits both ATP hydrolysis and ATP synthesis reactions. Moreover, we found that yaku´amide inhibitory activity is proton motive force (pmf) dependent, with lower inhibition in a more coupled system. Utilising cryo-EM, we obtained maps and models for the three main rotational states of murine ATP synthase (State 1 at 3.0 Å, 8 State 2 at 3.1 Å, and State 3 at 3.2 Å, overall). We observed several new features in our maps; however, we cannot definitively determine the exact mechanism of yaku amide’s inhibition on the protein due to either resolution limits or suboptimal binding of the inhibitor.}, author = {Lukic, Kristina}, issn = {2663-337X}, pages = {224}, publisher = {Institute of Science and Technology Austria}, title = {{Membrane proteins in plant physiology and bioenergetics : Investigating auxin efflux transporter PIN8 and ATP synthase inhibition by the novel inhibitor Yaku'amide B}}, doi = {10.15479/at:ista:17319}, year = {2024}, } @misc{14562, abstract = {Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. We have combined reverse genetics and cellular structural biology to describe how ArpC5 and ArpC5L differentially affect cell migration. Both define the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, affect protein dynamics and actin network ultrastructure. ArpC5 isoforms also affect the positioning of members of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongators, which mediate ArpC5 isoform–specific effects on the actin assembly level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling pathway enhancing cell migration. }, author = {Schur, Florian KM}, publisher = {Institute of Science and Technology Austria}, title = {{Research data of the publication "ArpC5 isoforms regulate Arp2/3 complex-dependent protrusion through differential Ena/VASP positioning"}}, doi = {10.15479/AT:ISTA:14562}, year = {2023}, } @unpublished{14644, abstract = {Transcription by RNA polymerase II (Pol II) can be repressed by noncoding RNA, including the human RNA Alu. However, the mechanism by which endogenous RNAs repress transcription remains unclear. Here we present cryo-electron microscopy structures of Pol II bound to Alu RNA, which reveal that Alu RNA mimics how DNA and RNA bind to Pol II during transcription elongation. Further, we show how domains of the general transcription factor TFIIF affect complex dynamics and control repressive activity. Together, we reveal how a non-coding RNA can regulate mammalian gene expression.}, author = {Tluckova, Katarina and Testa Salmazo, Anita P and Bernecky, Carrie A}, publisher = {Institute of Science and Technology Austria}, title = {{Mechanism of mammalian transcriptional repression by noncoding RNA}}, doi = {10.15479/AT:ISTA:14644}, year = {2023}, } @article{14719, abstract = {Lithium–sulfur batteries are regarded as an advantageous option for meeting the growing demand for high-energy-density storage, but their commercialization relies on solving the current limitations of both sulfur cathodes and lithium metal anodes. In this scenario, the implementation of lithium sulfide (Li2S) cathodes compatible with alternative anode materials such as silicon has the potential to alleviate the safety concerns associated with lithium metal. In this direction, here, we report a sulfur cathode based on Li2S nanocrystals grown on a catalytic host consisting of CoFeP nanoparticles supported on tubular carbon nitride. Nanosized Li2S is incorporated into the host by a scalable liquid infiltration–evaporation method. Theoretical calculations and experimental results demonstrate that the CoFeP–CN composite can boost the polysulfide adsorption/conversion reaction kinetics and strongly reduce the initial overpotential activation barrier by stretching the Li–S bonds of Li2S. Besides, the ultrasmall size of the Li2S particles in the Li2S–CoFeP–CN composite cathode facilitates the initial activation. Overall, the Li2S–CoFeP–CN electrodes exhibit a low activation barrier of 2.56 V, a high initial capacity of 991 mA h gLi2S–1, and outstanding cyclability with a small fading rate of 0.029% per cycle over 800 cycles. Moreover, Si/Li2S full cells are assembled using the nanostructured Li2S–CoFeP–CN cathode and a prelithiated anode based on graphite-supported silicon nanowires. These Si/Li2S cells demonstrate high initial discharge capacities above 900 mA h gLi2S–1 and good cyclability with a capacity fading rate of 0.28% per cycle over 150 cycles.}, author = {Mollania, Hamid and Zhang, Chaoqi and Du, Ruifeng and Qi, Xueqiang and Li, Junshan and Horta, Sharona and Ibáñez, Maria and Keller, Caroline and Chenevier, Pascale and Oloomi-Buygi, Majid and Cabot, Andreu}, issn = {1944-8252}, journal = {ACS Applied Materials and Interfaces}, number = {50}, pages = {58462–58475}, publisher = {American Chemical Society}, title = {{Nanostructured Li₂S cathodes for silicon-sulfur batteries}}, doi = {10.1021/acsami.3c14072}, volume = {15}, year = {2023}, } @article{12334, abstract = {Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. We have combined reverse genetics and cellular structural biology to describe how ArpC5 and ArpC5L differentially affect cell migration. Both define the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, affect protein dynamics and actin network ultrastructure. ArpC5 isoforms also affect the positioning of members of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongators, which mediate ArpC5 isoform–specific effects on the actin assembly level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling pathway enhancing cell migration.}, author = {Fäßler, Florian and Javoor, Manjunath and Datler, Julia and Döring, Hermann and Hofer, Florian and Dimchev, Georgi A and Hodirnau, Victor-Valentin and Faix, Jan and Rottner, Klemens and Schur, Florian KM}, issn = {2375-2548}, journal = {Science Advances}, keywords = {Multidisciplinary}, number = {3}, publisher = {American Association for the Advancement of Science}, title = {{ArpC5 isoforms regulate Arp2/3 complex–dependent protrusion through differential Ena/VASP positioning}}, doi = {10.1126/sciadv.add6495}, volume = {9}, year = {2023}, } @article{12759, abstract = {Stereological methods for estimating the 3D particle size and density from 2D projections are essential to many research fields. These methods are, however, prone to errors arising from undetected particle profiles due to sectioning and limited resolution, known as ‘lost caps’. A potential solution developed by Keiding, Jensen, and Ranek in 1972, which we refer to as the Keiding model, accounts for lost caps by quantifying the smallest detectable profile in terms of its limiting ‘cap angle’ (ϕ), a size-independent measure of a particle’s distance from the section surface. However, this simple solution has not been widely adopted nor tested. Rather, model-independent design-based stereological methods, which do not explicitly account for lost caps, have come to the fore. Here, we provide the first experimental validation of the Keiding model by comparing the size and density of particles estimated from 2D projections with direct measurement from 3D EM reconstructions of the same tissue. We applied the Keiding model to estimate the size and density of somata, nuclei and vesicles in the cerebellum of mice and rats, where high packing density can be problematic for design-based methods. Our analysis reveals a Gaussian distribution for ϕ rather than a single value. Nevertheless, curve fits of the Keiding model to the 2D diameter distribution accurately estimate the mean ϕ and 3D diameter distribution. While systematic testing using simulations revealed an upper limit to determining ϕ, our analysis shows that estimated ϕ can be used to determine the 3D particle density from the 2D density under a wide range of conditions, and this method is potentially more accurate than minimum-size-based lost-cap corrections and disector methods. Our results show the Keiding model provides an efficient means of accurately estimating the size and density of particles from 2D projections even under conditions of a high density.}, author = {Rothman, Jason Seth and Borges Merjane, Carolina and Holderith, Noemi and Jonas, Peter M and Angus Silver, R.}, issn = {1932-6203}, journal = {PLoS ONE}, number = {3 March}, publisher = {Public Library of Science}, title = {{Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy}}, doi = {10.1371/journal.pone.0277148}, volume = {18}, year = {2023}, } @article{12802, abstract = {Little is known about the critical metabolic changes that neural cells have to undergo during development and how temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), lead to autism, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different developmental stages. We found that the forebrain undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes, but what are the consequences of perturbing this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of LNAAs and lipids are interconnected in the cortex. Deletion of Slc7a5 in neurons affects the postnatal metabolic state, leading to a shift in lipid metabolism. Additionally, it causes stage- and cell-type-specific alterations in neuronal activity patterns, resulting in a long-term circuit dysfunction.}, author = {Knaus, Lisa and Basilico, Bernadette and Malzl, Daniel and Gerykova Bujalkova, Maria and Smogavec, Mateja and Schwarz, Lena A. and Gorkiewicz, Sarah and Amberg, Nicole and Pauler, Florian and Knittl-Frank, Christian and Tassinari, Marianna and Maulide, Nuno and Rülicke, Thomas and Menche, Jörg and Hippenmeyer, Simon and Novarino, Gaia}, issn = {0092-8674}, journal = {Cell}, keywords = {General Biochemistry, Genetics and Molecular Biology}, number = {9}, pages = {1950--1967.e25}, publisher = {Elsevier}, title = {{Large neutral amino acid levels tune perinatal neuronal excitability and survival}}, doi = {10.1016/j.cell.2023.02.037}, volume = {186}, year = {2023}, } @article{12832, abstract = {The development of cost-effective, high-activity and stable bifunctional catalysts for the oxygen reduction and evolution reactions (ORR/OER) is essential for zinc–air batteries (ZABs) to reach the market. Such catalysts must contain multiple adsorption/reaction sites to cope with the high demands of reversible oxygen electrodes. Herein, we propose a high entropy alloy (HEA) based on relatively abundant elements as a bifunctional ORR/OER catalyst. More specifically, we detail the synthesis of a CrMnFeCoNi HEA through a low-temperature solution-based approach. Such HEA displays superior OER performance with an overpotential of 265 mV at a current density of 10 mA/cm2, and a 37.9 mV/dec Tafel slope, well above the properties of a standard commercial catalyst based on RuO2. This high performance is partially explained by the presence of twinned defects, the incidence of large lattice distortions, and the electronic synergy between the different components, being Cr key to decreasing the energy barrier of the OER rate-determining step. CrMnFeCoNi also displays superior ORR performance with a half-potential of 0.78 V and an onset potential of 0.88 V, comparable with commercial Pt/C. The potential gap (Egap) between the OER overpotential and the ORR half-potential of CrMnFeCoNi is just 0.734 V. Taking advantage of these outstanding properties, ZABs are assembled using the CrMnFeCoNi HEA as air cathode and a zinc foil as the anode. The assembled cells provide an open-circuit voltage of 1.489 V, i.e. 90% of its theoretical limit (1.66 V), a peak power density of 116.5 mW/cm2, and a specific capacity of 836 mAh/g that stays stable for more than 10 days of continuous cycling, i.e. 720 cycles @ 8 mA/cm2 and 16.6 days of continuous cycling, i.e. 1200 cycles @ 5 mA/cm2.}, author = {He, Ren and Yang, Linlin and Zhang, Yu and Wang, Xiang and Lee, Seungho and Zhang, Ting and Li, Lingxiao and Liang, Zhifu and Chen, Jingwei and Li, Junshan and Ostovari Moghaddam, Ahmad and Llorca, Jordi and Ibáñez, Maria and Arbiol, Jordi and Xu, Ying and Cabot, Andreu}, issn = {2405-8297}, journal = {Energy Storage Materials}, number = {4}, pages = {287--298}, publisher = {Elsevier}, title = {{A CrMnFeCoNi high entropy alloy boosting oxygen evolution/reduction reactions and zinc-air battery performance}}, doi = {10.1016/j.ensm.2023.03.022}, volume = {58}, year = {2023}, } @phdthesis{12885, abstract = {High-performance semiconductors rely upon precise control of heat and charge transport. This can be achieved by precisely engineering defects in polycrystalline solids. There are multiple approaches to preparing such polycrystalline semiconductors, and the transformation of solution-processed colloidal nanoparticles is appealing because colloidal nanoparticles combine low cost with structural and compositional tunability along with rich surface chemistry. However, the multiple processes from nanoparticle synthesis to the final bulk nanocomposites are very complex. They involve nanoparticle purification, post-synthetic modifications, and finally consolidation (thermal treatments and densification). All these properties dictate the final material’s composition and microstructure, ultimately affecting its functional properties. This thesis explores the synthesis, surface chemistry and consolidation of colloidal semiconductor nanoparticles into dense solids. In particular, the transformations that take place during these processes, and their effect on the material’s transport properties are evaluated. }, author = {Calcabrini, Mariano}, isbn = {978-3-99078-028-2}, issn = {2663-337X}, pages = {82}, publisher = {Institute of Science and Technology Austria}, title = {{Nanoparticle-based semiconductor solids: From synthesis to consolidation}}, doi = {10.15479/at:ista:12885}, year = {2023}, } @phdthesis{13107, abstract = {Within the human body, the brain exhibits the highest rate of energy consumption amongst all organs, with the majority of generated ATP being utilized to sustain neuronal activity. Therefore, the metabolism of the mature cerebral cortex is geared towards preserving metabolic homeostasis whilst generating significant amounts of energy. This requires a precise interplay between diverse metabolic pathways, spanning from a tissue-wide scale to the level of individual neurons. Disturbances to this delicate metabolic equilibrium, such as those resulting from maternal malnutrition or mutations affecting metabolic enzymes, often result in neuropathological variants of neurodevelopment. For instance, mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), have been associated with autism and microcephaly. However, despite recent progress in the field, the extent of metabolic restructuring that occurs within the developing brain and the corresponding alterations in nutrient demands during various critical periods remain largely unknown. To investigate this, we performed metabolomic profiling of the murine cerebral cortex to characterize the metabolic state of the forebrain at different developmental stages. We found that the developing cortex undergoes substantial metabolic reprogramming, with specific sets of metabolites displaying stage-specific changes. According to our observations, we determined a distinct temporal period in postnatal development during which the cortex displays heightened reliance on LNAAs. Hence, using a conditional knock-out mouse model, we deleted Slc7a5 in neural cells, allowing us to monitor the impact of a perturbed neuronal metabolic state across multiple developmental stages of corticogenesis. We found that manipulating the levels of essential LNAAs in cortical neurons in vivo affects one particular perinatal developmental period critical for cortical network refinement. Abnormally low intracellular LNAA levels result in cell-autonomous alterations in neuronal lipid metabolism, excitability, and survival during this particular time window. Although most of the effects of Slc7a5 deletion on neuronal physiology are transient, derailment of these processes during this brief but crucial window leads to long-term circuit dysfunction in mice. In conclusion, out data indicate that the cerebral cortex undergoes significant metabolic reorganization during development. This process involves the intricate integration of multiple metabolic pathways to ensure optimal neuronal function throughout different developmental stages. Our findings offer a paradigm for understanding how neurons synchronize the expression of nutrient-related genes with their activity to allow proper brain maturation. Further, our results demonstrate that disruptions in these precisely calibrated metabolic processes during critical periods of brain development may result in neuropathological outcomes in mice and in humans.}, author = {Knaus, Lisa}, issn = {2663-337X}, pages = {147}, publisher = {Institute of Science and Technology Austria}, title = {{The metabolism of the developing brain : How large neutral amino acids modulate perinatal neuronal excitability and survival}}, doi = {10.15479/at:ista:13107}, year = {2023}, } @article{13202, abstract = {Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an essential role in neuronal activities through interaction with various proteins involved in signaling at membranes. However, the distribution pattern of PI(4,5)P2 and the association with these proteins on the neuronal cell membranes remain elusive. In this study, we established a method for visualizing PI(4,5)P2 by SDS-digested freeze-fracture replica labeling (SDS-FRL) to investigate the quantitative nanoscale distribution of PI(4,5)P2 in cryo-fixed brain. We demonstrate that PI(4,5)P2 forms tiny clusters with a mean size of ∼1000 nm2 rather than randomly distributed in cerebellar neuronal membranes in male C57BL/6J mice. These clusters show preferential accumulation in specific membrane compartments of different cell types, in particular, in Purkinje cell (PC) spines and granule cell (GC) presynaptic active zones. Furthermore, we revealed extensive association of PI(4,5)P2 with CaV2.1 and GIRK3 across different membrane compartments, whereas its association with mGluR1α was compartment specific. These results suggest that our SDS-FRL method provides valuable insights into the physiological functions of PI(4,5)P2 in neurons.}, author = {Eguchi, Kohgaku and Le Monnier, Elodie and Shigemoto, Ryuichi}, issn = {1529-2401}, journal = {The Journal of Neuroscience}, number = {23}, pages = {4197--4216}, publisher = {Society for Neuroscience}, title = {{Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons}}, doi = {10.1523/JNEUROSCI.1514-22.2023}, volume = {43}, year = {2023}, } @article{13968, abstract = {The use of multimodal readout mechanisms next to label-free real-time monitoring of biomolecular interactions can provide valuable insight into surface-based reaction mechanisms. To this end, the combination of an electrolyte-gated field-effect transistor (EG-FET) with a fiber optic-coupled surface plasmon resonance (FO-SPR) probe serving as gate electrode has been investigated to deconvolute surface mass and charge density variations associated to surface reactions. However, applying an electrochemical potential on such gold-coated FO-SPR gate electrodes can induce gradual morphological changes of the thin gold film, leading to an irreversible blue-shift of the SPR wavelength and a substantial signal drift. We show that mild annealing leads to optical and electronic signal stabilization (20-fold lower signal drift than as-sputtered fiber optic gates) and improved overall analytical performance characteristics. The thermal treatment prevents morphological changes of the thin gold-film occurring during operation, hence providing reliable and stable data immediately upon gate voltage application. Thus, the readout output of both transducing principles, the optical FO-SPR and electronic EG-FET, stays constant throughout the whole sensing time-window and the long-term effect of thermal treatment is also improved, providing stable signals even after 1 year of storage. Annealing should therefore be considered a necessary modification for applying fiber optic gate electrodes in real-time multimodal investigations of surface reactions at the solid-liquid interface.}, author = {Hasler, Roger and Steger-Polt, Marie Helene and Reiner-Rozman, Ciril and Fossati, Stefan and Lee, Seungho and Aspermair, Patrik and Kleber, Christoph and Ibáñez, Maria and Dostalek, Jakub and Knoll, Wolfgang}, issn = {2296-424X}, journal = {Frontiers in Physics}, publisher = {Frontiers}, title = {{Optical and electronic signal stabilization of plasmonic fiber optic gate electrodes: Towards improved real-time dual-mode biosensing}}, doi = {10.3389/fphy.2023.1202132}, volume = {11}, year = {2023}, } @article{14040, abstract = {Robust oxygenic photosynthesis requires a suite of accessory factors to ensure efficient assembly and repair of the oxygen-evolving photosystem two (PSII) complex. The highly conserved Ycf48 assembly factor binds to the newly synthesized D1 reaction center polypeptide and promotes the initial steps of PSII assembly, but its binding site is unclear. Here we use cryo-electron microscopy to determine the structure of a cyanobacterial PSII D1/D2 reaction center assembly complex with Ycf48 attached. Ycf48, a 7-bladed beta propeller, binds to the amino-acid residues of D1 that ultimately ligate the water-oxidising Mn4CaO5 cluster, thereby preventing the premature binding of Mn2+ and Ca2+ ions and protecting the site from damage. Interactions with D2 help explain how Ycf48 promotes assembly of the D1/D2 complex. Overall, our work provides valuable insights into the early stages of PSII assembly and the structural changes that create the binding site for the Mn4CaO5 cluster.}, author = {Zhao, Ziyu and Vercellino, Irene and Knoppová, Jana and Sobotka, Roman and Murray, James W. and Nixon, Peter J. and Sazanov, Leonid A and Komenda, Josef}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis}}, doi = {10.1038/s41467-023-40388-6}, volume = {14}, year = {2023}, }