Model-free characterization of topological edge and corner states in mechanical networks.

Topological materials can host edge and corner states that are protected from disorder and material imperfections. In particular, the topological edge states of mechanical structures present unmatched opportunities for achieving robust responses in wave guiding, sensing, computation, and filtering. However, determining whether a mechanical structure is topologically nontrivial and features topologically protected modes has hitherto relied on theoretical models. This strong requirement has limited the experimental and practical significance of topological mechanics to laboratory demonstrations. Here, we introduce and validate an experimental method to detect the topologically protected zero modes of mechanical structures without resorting to any modeling step. Our practical method is based on a simple electrostatic analogy: Topological zero modes are akin to electric charges. To detect them, we identify elementary mechanical molecules and measure their chiral polarization, a recently introduced marker of topology in chiral phases. Topological zero modes are then identified as singularities of the polarization field. Our method readily applies to any mechanical structure and effectively detects the edge and corner states of regular and higher-order topological insulators. Our findings extend the reach of chiral topological phases beyond designer materials and allow their direct experimental investigation.

Non-orientable order and non-commutative response in frustrated metamaterials.

From atomic crystals to animal flocks, the emergence of order in nature is captured by the concept of spontaneous symmetry breaking1-4. However, this cornerstone of physics is challenged when broken symmetry phases are frustrated by geometrical constraints. Such frustration dictates the behaviour of systems as diverse as spin ices5-8, confined colloidal suspensions9 and crumpled paper sheets10. These systems typically exhibit strongly degenerated and heterogeneous ground states and hence escape the Ginzburg-Landau paradigm of phase ordering. Here, combining experiments, simulations and theory we uncover an unanticipated form of topological order in globally frustrated matter: non-orientable order. We demonstrate this concept by designing globally frustrated metamaterials that spontaneously break a discrete [Formula: see text] symmetry. We observe that their equilibria are necessarily heteregeneous and extensively degenerated. We explain our observations by generalizing the theory of elasticity to non-orientable order-parameter bundles. We show that non-orientable equilibria are extensively degenerated due to the arbitrary location of topologically protected nodes and lines where the order parameter must vanish. We further show that non-orientable order applies more broadly to objects that are non-orientable themselves, such as buckled Möbius strips and Klein bottles. Finally, by applying time-dependent local perturbations to metamaterials with non-orientable order, we engineer topologically protected mechanical memories11-19, achieve non-commutative responses and show that they carry an imprint of the braiding of the loads' trajectories. Beyond mechanics, we envision non-orientability as a robust design principle for metamaterials that can effectively store information across scales, in fields as diverse as colloidal science8, photonics20, magnetism7 and atomic physics21.

The crystal structure of vaccinia virus protein E2 and perspectives on the prediction of novel viral protein folds.

The morphogenesis of vaccinia virus (VACV, family Poxviridae), the smallpox vaccine, is a complex process involving multiple distinct cellular membranes and resulting in multiple different forms of infectious virion. Efficient release of enveloped virions, which promote systemic spread of infection within hosts, requires the VACV protein E2 but the molecular basis of E2 function remains unclear and E2 lacks sequence homology to any well-characterised family of proteins. We solved the crystal structure of VACV E2 to 2.3 Å resolution, revealing that it comprises two domains with novel folds: an N-terminal annular (ring) domain and a C-terminal globular (head) domain. The C-terminal head domain displays weak structural homology with cellular (pseudo)kinases but lacks conserved surface residues or kinase features, suggesting that it is not enzymatically active, and possesses a large surface basic patch that might interact with phosphoinositide lipid headgroups. Recent deep learning methods have revolutionised our ability to predict the three-dimensional structures of proteins from primary sequence alone. VACV E2 is an exemplar 'difficult' viral protein target for structure prediction, being comprised of multiple novel domains and lacking sequence homologues outside Poxviridae. AlphaFold2 nonetheless succeeds in predicting the structures of the head and ring domains with high and moderate accuracy, respectively, allowing accurate inference of multiple structural properties. The advent of highly accurate virus structure prediction marks a step-change in structural virology and beckons a new era of structurally-informed molecular virology.

Disorder-Driven Quantum Transition in Relativistic Semimetals: Functional Renormalization via the Porous Medium Equation.

In the presence of randomness, a relativistic semimetal undergoes a quantum transition towards a diffusive phase. A standard approach relates this transition to the U(N) Gross-Neveu model in the limit of N→0. We show that the corresponding fixed point is infinitely unstable, demonstrating the necessity to include fluctuations beyond the usual Gaussian approximation. We develop a functional renormalization group method amenable to include these effects and show that the disorder distribution renormalizes following the so-called porous medium equation. We find that the transition is controlled by a nonanalytic fixed point drastically different from that of the U(N) Gross-Neveu model. Our approach provides a unique mechanism of spontaneous generation of a finite density of states and also characterizes the scaling behavior of the broad distribution of fluctuations close to the transition. It can be applied to other problems where nonanalytic effects may play a role, such as the Anderson localization transition.

Tagging of the vaccinia virus protein F13 with mCherry causes aberrant virion morphogenesis.

Vaccinia virus produces two distinct infectious virions; the single-enveloped intracellular mature virus (IMV), which remains in the cell until cell lysis, and the double-enveloped extracellular enveloped virus (EEV), which mediates virus spread. The latter is derived from a triple-enveloped intracellular enveloped virus (IEV) precursor, which is transported to the cell periphery by the kinesin-1 motor complex. This transport involves the viral protein A36 as well as F12 and E2. A36 is an integral membrane protein associated with the outer virus envelope and is the only known direct link between virion and kinesin-1 complex. Yet in the absence of A36 virion egress still occurs on microtubules, albeit at reduced efficiency. In this paper double-fluorescent labelling of the capsid protein A5 and outer-envelope protein F13 was exploited to visualize IEV transport by live-cell imaging in the absence of either A36 or F12. During the generation of recombinant viruses expressing both A5-GFP and F13-mCherry a plaque size defect was identified that was particularly severe in viruses lacking A36. Electron microscopy showed that this phenotype was caused by abnormal wrapping of IMV to form IEV, and this resulted in reduced virus egress to the cell surface. The aberrant wrapping phenotype suggests that the fluorescent fusion protein interferes with an interaction of F13 with the IMV surface that is required for tight association between IMVs and wrapping membranes. The severity of this defect suggests that these viruses are imperfect tools for characterizing virus egress.

Vaccinia virus egress mediated by virus protein A36 is reliant on the F12 protein.

Egress of vaccinia virus from its host cell is mediated by the microtubule-associated motor kinesin-1, and three viral proteins, A36 and the F12/E2 complex, have been implicated in this process. Deletion of F12 expression causes a more severe reduction in egress than deletion of A36 but whether these proteins are involved in the same or different mechanisms of kinesin-1 recruitment is unknown. Here it is shown that a virus lacking both proteins forms a smaller plaque than mutants lacking either gene alone, indicating non-redundant functions. A36 not only links virions directly to kinesin-1 but also nucleates actin polymerization to propel surface virions away from the host cell. To address the relative importance of these functions for virus spread, a panel of recombinant viruses was constructed in which the ability of A36 to bind kinesin-1 or to nucleate actin polymerization was abrogated individually or together, in the presence or absence of F12 expression. Analysis of these viruses revealed that in the presence of the F12 protein, loss of kinesin-1 interaction made a greater contribution to plaque size than did the formation of actin tails. However in the absence of F12, the ability of A36 to promote egress was abrogated. Therefore, the ability of A36 to promote egress by kinesin-1 is reliant on the F12 protein.

Vaccinia virus proteins A36 and F12/E2 show strong preferences for different kinesin light chain isoforms.

Vaccinia virus (VACV) utilizes microtubule-mediated trafficking at several stages of its life cycle, of which virus egress is the most intensely studied. During egress VACV proteins A36, F12 and E2 are involved in kinesin-1 interactions; however, the roles of these proteins remain poorly understood. A36 forms a direct link between virions and kinesin-1, yet in its absence VACV egress still occurs on microtubules. During a co-immunoprecipitation screen to seek an alternative link between virions and kinesin, A36 was found to bind isoform KLC1 rather than KLC2. The F12/E2 complex associates preferentially with the C-terminal tail of KLC2, to a region that overlaps the binding site of cellular 14-3-3 proteins. F12/E2 displaces 14-3-3 from KLC and, unlike 14-3-3, does not require phosphorylation of KLC for its binding. The region determining the KLC1 specificity of A36 was mapped to the KLC N-terminal heptad repeat region that is responsible for its association with kinesin heavy chain. Despite these differing binding properties F12/E2 can co-operatively enhance A36 association with KLC, particularly when using a KLC1-KLC2 chimaera that resembles several KLC1 spliceforms and can bind A36 and F12/E2 efficiently. This is the first example of a pathogen encoding multiple proteins that co-operatively associate with kinesin-1.

Topological index for periodically driven time-reversal invariant 2D systems.

We define a new Z2-valued index to characterize the topological properties of periodically driven two dimensional crystals when the time-reversal symmetry is enforced. This index is associated with a spectral gap of the evolution operator over one period of time. When two such gaps are present, the Kane-Mele index of the eigenstates with eigenvalues between the gaps is recovered as the difference of the gap indices. This leads to an expression for the Kane-Mele invariant in terms of the Wess-Zumino amplitude. We illustrate the relation of the new index to the edge states in finite geometries by numerically solving an explicit model on the square lattice that is periodically driven in a time-reversal invariant way.

Vaccinia virus protein complex F12/E2 interacts with kinesin light chain isoform 2 to engage the kinesin-1 motor complex.

During vaccinia virus morphogenesis, intracellular mature virus (IMV) particles are wrapped by a double lipid bilayer to form triple enveloped virions called intracellular enveloped virus (IEV). IEV are then transported to the cell surface where the outer IEV membrane fuses with the cell membrane to expose a double enveloped virion outside the cell. The F12, E2 and A36 proteins are involved in transport of IEVs to the cell surface. Deletion of the F12L or E2L genes causes a severe inhibition of IEV transport and a tiny plaque size. Deletion of the A36R gene leads to a smaller reduction in plaque size and less severe inhibition of IEV egress. The A36 protein is present in the outer membrane of IEVs, and over-expressed fragments of this protein interact with kinesin light chain (KLC). However, no interaction of F12 or E2 with the kinesin complex has been reported hitherto. Here the F12/E2 complex is shown to associate with kinesin-1 through an interaction of E2 with the C-terminal tail of KLC isoform 2, which varies considerably between different KLC isoforms. siRNA-mediated knockdown of KLC isoform 1 increased IEV transport to the cell surface and virus plaque size, suggesting interaction with KLC isoform 1 is somehow inhibitory of IEV transport. In contrast, knockdown of KLC isoform 2 did not affect IEV egress or plaque formation, indicating redundancy in virion egress pathways. Lastly, the enhancement of plaque size resulting from loss of KLC isoform 1 was abrogated by removal of KLC isoforms 1 and 2 simultaneously. These observations suggest redundancy in the mechanisms used for IEV egress, with involvement of KLC isoforms 1 and 2, and provide evidence of interaction of F12/E2 complex with the kinesin-1 complex.

Rabies virus envelope glycoprotein targets lentiviral vectors to the axonal retrograde pathway in motor neurons.

Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labeling techniques and confocal microscopy, we demonstrated that pseudotyping with rabies virus envelope glycoprotein (RV-G) enabled the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process revealed that these vectors trafficked through Rab5-positive endosomes and accumulated within a non-acidic Rab7 compartment. RV-G pseudotyped vectors were co-transported with both the tetanus neurotoxin-binding fragment and the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, and p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultured in compartmentalized chambers, we demonstrated that axonal retrograde transport of these vectors was rapid and efficient; however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, which suggests a novel area for improvement of gene therapy vectors.

Magnetic dephasing in mesoscopic spin glasses.

We have measured universal conductance fluctuations in the metallic spin glass Ag:Mn as a function of temperature and magnetic field. From this measurement, we can access the phase coherence time of the electrons in the spin glass. We show that this phase coherence time increases with both the inverse of the temperature and the magnetic field. From this, we deduce that decoherence mechanisms are still active even deep in the spin glass phase.

Transport and stability of the vaccinia virus A34 protein is affected by the A33 protein.

Vaccinia virus (VACV) has two infectious forms called intracellular mature virus and extracellular enveloped virus (EEV). Two of the seven viral proteins in the EEV outer envelope, A33 and A34, are type II membrane glycoproteins that each interact with another EEV protein called B5; however, evidence for direct A33-A34 interaction is lacking. The localization and stability of A34 is affected by B5 and here data are presented showing that A34 is also affected by A33. In the absence of A33, just as without B5, the level, localization and glycosylation profile of A34 was altered. However, the glycosylation profile of A34 without A33 is different to that observed in the absence of B5, and A34 accumulates in the Golgi apparatus rather than in the endoplasmic reticulum. Thus, A34 requires more than one other EEV protein for its processing and cellular transport.

Improved expression of secreted and membrane-targeted proteins in insect cells.

Secretory and membrane-bound proteins are generally produced in lower amounts in insect cells compared with cytoplasmic and nuclear proteins. There may be many reasons for this, including degradation of recombinant proteins by proteases, competition for cellular resources between native and recombinant proteins, and physical blockage of the secretory pathways. In the present study, we describe the construction of a baculovirus in which chiA (chitinase) and cath (cathepsin) genes have been deleted and show improved recombinant protein expression using this vector. We confirmed the complete removal of both genes by PCR, restriction enzyme analysis and enzyme assays, and the modified virus DNA was shown to be stable in bacterial cells over multiple passages. A selection of recombinant genes were inserted into the double-deletion virus and their expression levels compared with recombinant viruses that had single or no gene deletions. In all instances, the double-deletion viruses showed greatly enhanced levels of protein production for both secreted and nuclear/cytoplasmic proteins. In summary, we have conclusively demonstrated the importance of this deletion vector for the high-level production of recombinant proteins.

Vaccinia protein F12 has structural similarity to kinesin light chain and contains a motor binding motif required for virion export.

Vaccinia virus (VACV) uses microtubules for export of virions to the cell surface and this process requires the viral protein F12. Here we show that F12 has structural similarity to kinesin light chain (KLC), a subunit of the kinesin-1 motor that binds cargo. F12 and KLC share similar size, pI, hydropathy and cargo-binding tetratricopeptide repeats (TPRs). Moreover, molecular modeling of F12 TPRs upon the crystal structure of KLC2 TPRs showed a striking conservation of structure. We also identified multiple TPRs in VACV proteins E2 and A36. Data presented demonstrate that F12 is critical for recruitment of kinesin-1 to virions and that a conserved tryptophan and aspartic acid (WD) motif, which is conserved in the kinesin-1-binding sequence (KBS) of the neuronal protein calsyntenin/alcadein and several other cellular kinesin-1 binding proteins, is essential for kinesin-1 recruitment and virion transport. In contrast, mutation of WD motifs in protein A36 revealed they were not required for kinesin-1 recruitment or IEV transport. This report of a viral KLC-like protein containing a KBS that is conserved in several cellular proteins advances our understanding of how VACV recruits the kinesin motor to virions, and exemplifies how viruses use molecular mimicry of cellular components to their advantage.

Measuring overlaps in mesoscopic spin glasses via conductance fluctuations.

We consider the electronic transport in a mesoscopic metallic spin glass. We show that the distribution of overlaps between spin configurations can be inferred from the reduction of the conductance fluctuations by the magnetic impurities. Using this property, we propose new experimental protocols to probe spin glasses directly through their overlaps.

The baculovirus P10 protein of Autographa californica nucleopolyhedrovirus forms two distinct cytoskeletal-like structures and associates with polyhedral occlusion bodies during infection.

The role of the microtubule-associated P10 protein of baculoviruses is not yet understood. P10 has previously been linked with the formation of a number of cytoskeletal-like or cytoskeleton-associated structures in the nucleus and cytoplasm, thought to be involved in the morphogenesis of virus polyhedral occlusion bodies. The formation of these structures was studied by immunofluorescence laser scanning confocal microscopy in TN368 cells, a model system amenable to the study of virus interaction with the host cell cytoskeleton. We show that the Autographa californica nucleopolyhedrovirus P10 protein forms two distinct cytoskeletal-like structures, microtubule-associated filaments and perinuclear tubular aggregates. P10 also associates with polyhedral occlusion bodies. Depolymerisation of the microtubule network with colchicine prevents formation of P10 filaments but not of P10 tubules. Colchicine treatment enhances the association of P10 with occlusion bodies. Transient expression of P10 showed that both filaments and tubules can form in the absence of other viral proteins. We postulate a number of possible roles of the P10 protein during virus infection and morphogenesis.

Random quantum Ising chains with competing interactions.

In this paper we discuss the criticality of a quantum Ising spin chain with competing random ferromagnetic and antiferromagnetic couplings. Quantum fluctuations are introduced via random local transverse fields. First we consider the chain with couplings between first and second neighbors only and then generalize the study to a quantum analog of the Viana-Bray model, defined on a small world random lattice. We use the Dasgupta-Ma decimation technique, both analytically and numerically, and focus on the scaling of the lattice topology, whose determination is necessary to define any infinite disorder transition beyond the chain. In the first case, at the transition the model renormalizes towards the chain, with the infinite disorder fixed point described by Fisher. This corresponds to the irrelevance of the competition induced by the second neighbors couplings. As opposed to this case, this infinite disorder transition is found to be unstable towards the introduction of an arbitrary small density of long range couplings in the small world models.

Inducible gene silencing in podocytes: a new tool for studying glomerular function.

Glomerular filtration is one of the primary functions of the kidney. Podocytes, a highly specialized cell type found in glomeruli, are believed to play a critical role in that function. Null mutations of genes expressed in podocytes like WT1, nephrin, and NEPH1 result in an embryo and perinatal lethal phenotype and therefore do not allow the functional analysis of these genes in the adult kidney. Here is describes the generation of a model that will allow such studies. We have engineered transgenic mice in which the disruption of targeted genes can be induced in a temporally controlled fashion in podocytes. For this, a transgene encoding the mutated estrogen receptor-Cre recombinase fusion protein was introduced into the mouse genome. Animals were crossed with Z/AP reporter mice to test for efficient and inducible recombination. We found that, after injection of inducer drug tamoxifen, Cre fusion protein translocates to the nuclei of podocytes, where it becomes active and mediates recombination of DNA carrying loxP target sequences. These animals provide for the first time a tool for silencing genes selectively in podocytes of adult animals.