Reversed Phase-Liquid Chromatography for Recombinant AAV Genome Integrity Assessment.

After decades of research, gene therapy products have reached market maturity in recent years. Recombinant adeno-associated viruses (rAAVs) are one of the most promising gene delivery vehicles and are currently under intense scientific investigation. These next-generation medicines remain very challenging when it comes to designing appropriate analytical techniques for quality control. One critical quality attribute is the integrity of ssDNA incorporated in these vectors. The genome is the active compound driving rAAV therapy and therefore requires proper assessment and quality control. Current techniques for rAAV genome characterization include next-generation sequencing, quantitative polymerase chain reaction, analytical ultracentrifugation (AUC), and capillary gel electrophoresis (CGE), yet each of them presents their limitations or lack of user-friendliness. In this work, we demonstrate for the first time the potential of ion pairing-reverse phase-liquid chromatography (IP-RP-LC) to characterize the integrity of rAAV genomes. The obtained results were supported by two orthogonal techniques, AUC and CGE. IP-RP-LC can be performed above DNA melting temperatures, avoiding the detection of secondary DNA isoforms, and does not require the use of dyes due to UV detection. We demonstrate that this technique is suitable for batch comparability, different rAAV serotypes (AAV2 and AAV8), internal vs external (inside vs outside the capsid) DNA analysis, and contaminated samples. Overall, it is exceptionally user-friendly, needs limited sample preparation, has high reproducibility, and permits fractionation for further peak characterization. All of these factors add significant value of IP-RP-LC to the analytical toolbox of rAAV genome assessment.

Genuine Many-Body Quantum Scars along Unstable Modes in Bose-Hubbard Systems.

The notion of many-body quantum scars is associated with special eigenstates, usually concentrated in certain parts of Hilbert space, that give rise to robust persistent oscillations in a regime that globally exhibits thermalization. Here we extend these studies to many-body systems possessing a true classical limit characterized by a high-dimensional chaotic phase space, which are not subject to any particular dynamical constraint. We demonstrate genuine quantum scarring of wave functions concentrated in the vicinity of unstable classical periodic mean-field modes in the paradigmatic Bose-Hubbard model. These peculiar quantum many-body states exhibit distinct phase-space localization about those classical modes. Their existence is consistent with Heller's scar criterion and appears to persist in the thermodynamic long-lattice limit. Launching quantum wave packets along such scars leads to observable long-lasting oscillations, featuring periods that scale asymptotically with classical Lyapunov exponents, and displaying intrinsic irregularities that reflect the underlying chaotic dynamics, as opposed to regular tunnel oscillations.

Controlling Quantum Chaos: Optimal Coherent Targeting.

One of the principal goals of controlling classical chaotic dynamical systems is known as targeting, which is the very weakly perturbative process of using the system's extreme sensitivity to initial conditions in order to arrive at a predetermined target state. It is shown that a generalization to chaotic quantum systems is possible in the semiclassical regime, but requires tailored perturbations whose effects must undo the dynamical spreading of the evolving quantum state. The procedure described here is applied to initially minimum uncertainty wave packets in the quantum kicked rotor, a preeminent quantum chaotic paradigm, to illustrate the method, and investigate its accuracy. The method's error can be made to vanish as ℏ→0.

Matrix-independent boron isotope analysis of silicate and carbonate reference materials by ultraviolet femtosecond laser ablation multi-collector inductively coupled plasma mass spectrometry with application to the cold-water coral Desmophyllum dianthus.

RATIONALE: Boron isotopes are a powerful tool for pH reconstruction in marine carbonates and as a tracer for fluid-mineral interaction in geochemistry. Microanalytical approaches based on laser ablation multi-collector inductively coupled plasma mass spectrometry (LA-MC-ICP-MS) often suffer from effects induced by the sample matrix. In this study, we investigate matrix-independent analyses of B isotopic ratios and apply this technique to cold-water corals. METHODS: We employ a customized 193 nm femtosecond laser ablation system (Solstice, Spectra-Physics) coupled to a MC-ICP-MS system (Nu Plasma II, Nu Instruments) equipped with electron multipliers for in situ measurements of B isotopic ratios (11 B/10 B) at the micrometric scale. We analyzed various reference materials of silicate and carbonate matrices using non-matrix matched calibration without employing any correction. This approach was then applied to investigate defined increments in coral samples from a Chilean fjord. RESULTS: We obtained accurate B isotopic ratios with a reproducibility of ±0.9‰ (2 SD) for various reference materials including silicate glasses (GOR132-G, StHs6/80-G, ATHO-G and NIST SRM 612), clay (IAEA-B-8) and carbonate (JCp-1) using the silicate glass NIST SRM 610 as calibration standard, which shows that neither laser-induced nor ICP-related matrix effects are detectable. The application to cold-water corals (Desmophyllum dianthus) reveals minor intra-skeleton variations in δ11 B with average values between 23.01‰ and 25.86‰. CONCLUSIONS: Our instrumental set-up provides accurate and precise B isotopic ratios independently of the sample matrix at the micrometric scale. This approach opens a wide field of application in geochemistry, including pH reconstruction in biogenic carbonates and deciphering processes related to fluid-mineral interaction.

Quantitative Trait Locus Mapping for Fire Blight Resistance in an F2 Population of Malus fusca MAL0045 Uncovers Novel Resistance Loci.

Several fire blight resistance loci in Malus genotypes map on different linkage groups (LGs) representing chromosomes of the domesticated apple. Prior genetics studies primarily focused on F1 populations. A strong resistance quantitative trait locus (QTL) explained up to 66% of phenotypic variance in an F1 progeny derived from crossing the highly resistant wild apple genotype Malus fusca MAL0045 and the highly susceptible apple cultivar 'Idared', which was previously mapped on LG10 (Mfu10) of MAL0045. Strains of the causative bacterial pathogen Erwinia amylovora, notably those that show a single nucleotide polymorphism in the avrRpt2EA effector protein sequence at position 156 (e.g., Ea3049), are more virulent and overcome some known fire blight resistance donors and their QTLs. However, MAL0045 is resistant to Ea3049 and Mfu10 is not overcome, but most of the F1 progeny were highly susceptible to this strain. This phenomenon led to the assumption that other putative resistance factors not segregating in the F1 progeny might be present in the genome of MAL0045. Here, we crossed F1 progeny together to obtain 135 F2 individuals. Facilitated by genotyping-by-sequencing and phenotypic assessments, we identified and mapped two novel resistance QTLs in these F2 individuals on LGs 4 and 15, which were not identified in the F1. To our knowledge, these are the first resistance QTLs mapped in F2 progeny in Malus. In addition, we report that neither MAL0045 nor Mfu10 is broken down by a highly aggressive U.S. strain, LA635, after analyses in the original F1 individuals. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin.

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirm this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin, and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics.

Binding Mode Characterization of Osteopontin on Hydroxyapatite by Solution NMR Spectroscopy.

Extracellular matrix glycoproteins play a major role in bone mineralization and modulation of osteogenesis. Among these, the intrinsically disordered protein osteopontin (OPN) is associated with the inhibition of formation, growth and proliferation of the bone mineral hydroxyapatite (HAP). Furthermore, post-translational modifications like phosphorylation can alter conformations and interaction properties of intrinsically disordered proteins (IDPs). Therefore, the actual interaction of OPN with a HAP surface on an atomic level and how this interaction is affected by phosphorylation is of great interest. Here, we study the interaction of full-length OPN on the surface of suspended HAP nanoparticles by solution NMR spectroscopy. We report the binding modes of this IDP and provide evidence for the influence of hyperphosphorylation on the binding character and an explanation for the differing roles in biomineralization. Our study moreover presents an easy and suitable option to measure interaction of nanoparticles in a stable suspension with full-length proteins.

Emergence of a Renormalized 1/N Expansion in Quenched Critical Many-Body Systems.

We consider the fate of 1/N expansions in unstable many-body quantum systems, as realized by a quench across criticality, and show the emergence of e^{2λt}/N as a renormalized parameter ruling the quantum-classical transition and accounting nonperturbatively for the local divergence rate λ of mean-field solutions. In terms of e^{2λt}/N, quasiclassical expansions of paradigmatic examples of criticality, like the self-trapping transition in an integrable Bose-Hubbard dimer and the generic instability of attractive bosonic systems toward soliton formation, are pushed to arbitrarily high orders. The agreement with numerical simulations supports the general nature of our results in the appropriately combined long-time λt→∞ quasiclassical N→∞ regime, out of reach of expansions in the bare parameter 1/N. For scrambling in many-body hyperbolic systems, our results provide formal grounds to a conjectured multiexponential form of out-of-time-ordered correlators.

Two-Dimensional-Dirac Surface States and Bulk Gap Probed via Quantum Capacitance in a Three-Dimensional Topological Insulator.

BiSbTeSe2 is a 3D topological insulator (3D-TI) with Dirac type surface states and low bulk carrier density, as donors and acceptors compensate each other. Dominating low-temperature surface transport in this material is heralded by Shubnikov-de Haas oscillations and the quantum Hall effect. Here, we experimentally probe and model the electronic density of states (DOS) in thin layers of BiSbTeSe2 by capacitance experiments both without and in quantizing magnetic fields. By probing the lowest Landau levels, we show that a large fraction of the electrons filled via field effect into the system ends up in (localized) bulk states and appears as a background DOS. The surprisingly strong temperature dependence of such background DOS can be traced back to Coulomb interactions. Our results point at the coexistence and intimate coupling of Dirac surface states with a bulk many-body phase (a Coulomb glass) in 3D-TIs.

Gate-Tunable Two-Dimensional Superlattices in Graphene.

We report an efficient technique to induce gate-tunable two-dimensional superlattices in graphene by the combined action of a back gate and a few-layer graphene patterned bottom gate complementary to existing methods. The patterned gates in our approach can be easily fabricated and implemented in van der Waals stacking procedures, allowing flexible use of superlattices with arbitrary geometry. In transport measurements on a superlattice with a lattice constant a = 40 nm, well-pronounced satellite Dirac points and signatures of the Hofstadter butterfly including a nonmonotonic quantum Hall response are observed. Furthermore, the experimental results are accurately reproduced in transport simulations and show good agreement with features in the calculated band structure. Overall, we present a comprehensive picture of graphene-based superlattices, featuring a broad range of miniband effects, both in experiment and in theoretical modeling. The presented technique is suitable for studying more advanced geometries which are not accessible by other methods.

Spin Relaxation in s-Wave Superconductors in the Presence of Resonant Spin-Flip Scatterers.

Employing analytical methods and quantum transport simulations we investigate the relaxation of quasiparticle spins in graphene proximitized by an s-wave superconductor in the presence of resonant magnetic and spin-orbit active impurities. Off resonance, the relaxation increases with decreasing temperature when electrons scatter off magnetic impurities-the Hebel-Slichter effect-and decreases when impurities have spin-orbit coupling. This distinct temperature dependence (not present in the normal state) uniquely discriminates between the two scattering mechanisms. However, we show that the Hebel-Slichter picture breaks down at resonances. The emergence of Yu-Shiba-Rusinov bound states within the superconducting gap redistributes the spectral weight away from magnetic resonances. The result is opposite to the Hebel-Slichter expectation: the spin relaxation decreases with decreasing temperature. Our findings hold for generic s-wave superconductors with resonant magnetic impurities, but also, as we show, for resonant magnetic Josephson junctions.

Magnetoconductance, Quantum Hall Effect, and Coulomb Blockade in Topological Insulator Nanocones.

Magnetotransport through cylindrical topological insulator (TI) nanowires is governed by the interplay between quantum confinement and geometric (Aharonov-Bohm and Berry) phases. Here, we argue that the much broader class of TI nanowires with varying radius-for which a homogeneous coaxial magnetic field induces a varying Aharonov-Bohm flux that gives rise to a nontrivial masslike potential along the wire-is accessible by studying its simplest member, a TI nanocone. Such nanocones allow us to observe intriguing mesoscopic transport phenomena: While the conductance in a perpendicular magnetic field is quantized due to higher-order topological hinge states, it shows resonant transmission through Dirac Landau levels in a coaxial magnetic field. Furthermore, it may act as a quantum magnetic bottle, confining surface Dirac electrons and leading to a largely interaction-dominated regime of Coulomb blockade type. We show numerically that the above-mentioned effects occur for experimentally accessible values of system size and magnetic field, suggesting that TI nanocone junctions may serve as building blocks for Dirac electron optics setups.

Anomalous Cyclotron Motion in Graphene Superlattice Cavities.

We consider graphene superlattice miniband fermions probed by electronic interferometry in magnetotransport experiments. By decoding the observed Fabry-Pérot interference patterns together with our corresponding quantum transport simulations, we find that the Dirac quasiparticles originating from the superlattice minibands do not undergo conventional cyclotron motion but follow more subtle trajectories. In particular, dynamics at low magnetic fields is characterized by peculiar, straight trajectory segments. Our results provide new insights into superlattice miniband fermions and open up novel possibilities to use periodic potentials in electron optics experiments.

Modulated Scanning Fluorimetry Can Quickly Assess Thermal Protein Unfolding Reversibility in Microvolume Samples.

Determining the temperature at which the thermal unfolding of a protein starts becoming irreversible is relevant for many areas of protein research. Until now, published methods cannot determine, within a reasonable time frame and with moderate sample consumption, the exposure temperature that starts causing irreversible protein unfolding. We present modulated scanning fluorimetry (MSF) and share a software (MSF Analyzer), which can be used to derive nonreversibility curves of thermal protein unfolding from a series of incremental temperature cycles performed on only 10 µL samples, consuming as low as a few micrograms of protein. Further processing of the data can yield the onset temperature that starts causing nonreversible protein unfolding. The MSF method is based on the hardware of the already existing nanoDSF technology and can be applied to dozens of samples simultaneously. Here, we use MSF to study how solution pH affects the reversibility of thermal protein unfolding of several model proteins to show that the nonreversibility onset temperature (Tnr) is a unique biophysical parameter, providing orthogonal information from thermal protein denaturation data and insights into the validity of thermal unfolding analysis in the context of equilibrium thermodynamics. We also show that MSF can be used to study enzyme stability after exposure to high temperatures. Besides, we demonstrate that protein thermal unfolding and nonreversibility can be affected in different ways upon modifications like PEG-ylation or labeling with fluorescent dyes. Finally, we show that MSF can be used to study the effect of various protein interactions on thermal protein unfolding reversibility. With the diverse examples in this work, we reveal how MSF can provide orthogonal information from thermal denaturation experiments that can bring benefits to various areas of protein research. The MSF Analyzer software is available at https://github.com/CoriolisPharmaResearch/MSFAnalyser.

Conformational switching of the molecular chaperone Hsp90 via regulated phosphorylation.

Hsp90 is an essential molecular chaperone in the eukaryotic cytosol. Its function is modulated by cochaperones and posttranslational modifications. Importantly, the phosphatase Ppt1 is a dedicated regulator of the Hsp90 chaperone system. Little is known about Ppt1-dependent phosphorylation sites and how these affect Hsp90 activity. Here, we identified the major phosphorylation sites of yeast Hsp90 in its middle or the C-terminal domain and determined the subset regulated by Ppt1. In general, phosphorylation decelerates the Hsp90 machinery, reduces chaperone function in vivo, sensitizes yeast cells to Hsp90 inhibition and affects DNA repair processes. Modification of one particular site (S485) is lethal, whereas others modulate Hsp90 activity via distinct mechanisms affecting the ATPase activity, cochaperone binding and manipulating conformational transitions in Hsp90. Our mechanistic analysis reveals that phosphorylation of Hsp90 permits a regulation of the conformational cycle at distinct steps by targeting switch points for the communication of remote regions within Hsp90.

Regulation of the Hsp90 system.

Hsp90 is a highly conserved and abundant chaperone. It participates in essential cellular activities by supporting the maturation process of its client proteins, many of which are protein kinases and steroid receptors. Client processing is achieved via extensive conformational changes within the dimeric chaperone. This requires an ATP hydrolysis activity that is controlled by auto-inhibitory mechanisms and several structurally diverse cofactors. Especially the client-specificity of Hsp90 depends on client-specific cofactors, which can adapt Hsp90's activities to the client requirements at different conditions and in different cell types. Additionally, post-translational modifications can influence almost every aspect of Hsp90's interactions and activities. In this review, we present these regulatory principles, discuss the factors that have an impact on Hsp90's function and elaborate the mechanisms that are responsible for regulating the Hsp90 machinery.

Mapping of fire blight resistance in Malus ×robusta 5 flowers following artificial inoculation.

BACKGROUND: Although the most common path of infection for fire blight, a severe bacterial disease on apple, is via host plant flowers, quantitative trait loci (QTLs) for fire blight resistance to date have exclusively been mapped following shoot inoculation. It is not known whether the same mechanism underlies flower and shoot resistance. RESULTS: We report the detection of a fire blight resistance QTL following independent artificial inoculation of flowers and shoots on two F1 segregating populations derived from crossing resistant Malus ×robusta 5 (Mr5) with susceptible 'Idared' and 'Royal Gala' in experimental orchards in Germany and New Zealand, respectively. QTL mapping of phenotypic datasets from artificial flower inoculation of the 'Idared' × Mr5 population with Erwinia amylovora over several years, and of the 'Royal Gala' × Mr5 population in a single year, revealed a single major QTL controlling floral fire blight resistance on linkage group 3 (LG3) of Mr5. This QTL corresponds to the QTL on LG3 reported previously for the 'Idared' × Mr5 and an 'M9' × Mr5 population following shoot inoculation in the glasshouse. Interval mapping of phenotypic data from shoot inoculations of subsets from both flower resistance populations re-confirmed that the resistance QTL is in the same position on LG3 of Mr5 as that for flower inoculation. These results provide strong evidence that fire blight resistance in Mr5 is controlled by a major QTL on LG3, independently of the mode of infection, rootstock and environment. CONCLUSIONS: This study demonstrates for the first time that resistance to fire blight caused by Erwinia amylovora is independent of the mode of inoculation at least in Malus ×robusta 5.

Partial Fermionization: Spectral Universality in 1D Repulsive Bose Gases.

Because of the vast growth of the many-body level density with excitation energy, its smoothed form is of central relevance for spectral and thermodynamic properties of interacting quantum systems. We compute the cumulative of this level density for confined one-dimensional continuous systems with repulsive short-range interactions. We show that the crossover from an ideal Bose gas to the strongly correlated, fermionized gas, i.e., partial fermionization, exhibits universal behavior: Systems with very few and up to many particles share the same underlying spectral features. In our derivation we supplement quantum cluster expansions with short-time dynamical information. Our nonperturbative analytical results are in excellent agreement with numerics for systems of experimental relevance in cold atom physics, such as interacting bosons on a ring (Lieb-Liniger model) or subject to harmonic confinement. Our method provides predictions for excitation spectra that enable access to finite-temperature thermodynamics in large parameter ranges.

Reversible Quantum Information Spreading in Many-Body Systems near Criticality.

Quantum chaotic interacting N-particle systems are assumed to show fast and irreversible spreading of quantum information on short (Ehrenfest) time scales ∼logN. Here, we show that, near criticality, certain many-body systems exhibit fast initial scrambling, followed subsequently by oscillatory behavior between reentrant localization and delocalization of information in Hilbert space. We consider both integrable and nonintegrable quantum critical bosonic systems with attractive contact interaction that exhibit locally unstable dynamics in the corresponding many-body phase space of the large-N limit. Semiclassical quantization of the latter accounts for many-body correlations in excellent agreement with simulations. Most notably, it predicts an asymptotically constant local level spacing ℏ/τ, again given by τ∼logN. This unique timescale governs the long-time behavior of out-of-time-order correlators that feature quasiperiodic recurrences indicating reversibility.

Enhancement of Many-Body Quantum Interference in Chaotic Bosonic Systems: The Role of Symmetry and Dynamics.

Although highly successful, the truncated Wigner approximation (TWA) leaves out many-body quantum interference between mean-field Gross-Pitaevskii solutions as well as other quantum effects, and is therefore essentially classical. Turned around, if a system's quantum properties deviate from TWA, they must be exhibiting some quantum phenomenon, such as localization, diffraction, or tunneling. Here, we examine a particular interference effect arising from discrete symmetries, which can significantly enhance quantum observables with respect to the TWA prediction, and derive an augmented TWA in order to incorporate them. Using the Bose-Hubbard model for illustration, we further show strong evidence for the presence of dynamical localization due to remaining differences between the TWA predictions and quantum results.