Observation of gauge invariance in a 71-site Bose-Hubbard quantum simulator.

The modern description of elementary particles, as formulated in the standard model of particle physics, is built on gauge theories1. Gauge theories implement fundamental laws of physics by local symmetry constraints. For example, in quantum electrodynamics Gauss's law introduces an intrinsic local relation between charged matter and electromagnetic fields, which protects many salient physical properties, including massless photons and a long-ranged Coulomb law. Solving gauge theories using classical computers is an extremely arduous task2, which has stimulated an effort to simulate gauge-theory dynamics in microscopically engineered quantum devices3-6. Previous achievements implemented density-dependent Peierls phases without defining a local symmetry7,8, realized mappings onto effective models to integrate out either matter or electric fields9-12, or were limited to very small systems13-16. However, the essential gauge symmetry has not been observed experimentally. Here we report the quantum simulation of an extended U(1) lattice gauge theory, and experimentally quantify the gauge invariance in a many-body system comprising matter and gauge fields. These fields are realized in defect-free arrays of bosonic atoms in an optical superlattice of 71 sites. We demonstrate full tunability of the model parameters and benchmark the matter-gauge interactions by sweeping across a quantum phase transition. Using high-fidelity manipulation techniques, we measure the degree to which Gauss's law is violated by extracting probabilities of locally gauge-invariant states from correlated atom occupations. Our work provides a way to explore gauge symmetry in the interplay of fundamental particles using controllable large-scale quantum simulators.

Thermalization of Gauge Theories from their Entanglement Spectrum.

Using dual theories embedded into a larger unphysical Hilbert space along entanglement cuts, we study the entanglement structure of Z_{2} lattice gauge theory in (2+1) spacetime dimensions. We demonstrate Li and Haldane's conjecture, and show consistency of the entanglement Hamiltonian with the Bisognano-Wichmann theorem. Studying nonequilibrium dynamics after a quench, we provide an extensive description of thermalization in Z_{2} gauge theory which proceeds in a characteristic sequence: Maximization of the Schmidt rank and spreading of level repulsion at early times, self-similar evolution with scaling coefficients α=0.8±0.2 and ß=0.0±0.1 at intermediate times, and finally thermal saturation of the von Neumann entropy.

Thermalization dynamics of a gauge theory on a quantum simulator.

Gauge theories form the foundation of modern physics, with applications ranging from elementary particle physics and early-universe cosmology to condensed matter systems. We perform quantum simulations of the unitary dynamics of a U(1) symmetric gauge field theory and demonstrate emergent irreversible behavior. The highly constrained gauge theory dynamics are encoded in a one-dimensional Bose-Hubbard simulator, which couples fermionic matter fields through dynamical gauge fields. We investigated global quantum quenches and the equilibration to a steady state well approximated by a thermal ensemble. Our work may enable the investigation of elusive phenomena, such as Schwinger pair production and string breaking, and paves the way for simulating more complex, higher-dimensional gauge theories on quantum synthetic matter devices.

An optimized 3D-printed perfusion bioreactor for homogeneous cell seeding in bone substitute scaffolds for future chairside applications.

A clinical implementation of cell-based bone regeneration in combination with scaffold materials requires the development of efficient, controlled and reproducible seeding procedures and a tailor-made bioreactor design. A perfusion system for efficient, homogeneous, and rapid seeding with human adipogenic stem cells in bone substitute scaffolds was designed. Variants concerning medium inlet and outlet port geometry, i.e. cylindrical or conical diffuser, cell concentration, perfusion mode and perfusion rates were simulated in silico. Cell distribution during perfusion was monitored by dynamic [18F]FDG micro-PET/CT and validated by laser scanning microscopy with three-dimensional image reconstruction. By iterative feedback of the in silico and in vitro experiments, the homogeneity of cell distribution throughout the scaffold was optimized with adjustment of flow rates, cell density and perfusion properties. Finally, a bioreactor with a conical diffusor geometry was developed, that allows a homogeneous cell seeding (hoover coefficient: 0.24) in less than 60 min with an oscillating perfusion mode. During this short period of time, the cells initially adhere within the entire scaffold and stay viable. After two weeks, the formation of several cell layers was observed, which was associated with an osteogenic differentiation process. This newly designed bioreactor may be considered as a prototype for chairside application.

Insights into hRPA32 C-terminal domain--mediated assembly of the simian virus 40 replisome.

Simian virus 40 (SV40) provides a model system for the study of eukaryotic DNA replication, in which the viral protein, large T antigen (Tag), marshals human proteins to replicate the viral minichromosome. SV40 replication requires interaction of Tag with the host single-stranded DNA-binding protein, replication protein A (hRPA). The C-terminal domain of the hRPA32 subunit (RPA32C) facilitates initiation of replication, but whether it interacts with Tag is not known. Affinity chromatography and NMR revealed physical interaction between hRPA32C and the Tag origin DNA-binding domain, and a structural model of the complex was determined. Point mutations were then designed to reverse charges in the binding sites, resulting in substantially reduced binding affinity. Corresponding mutations introduced into intact hRPA impaired initiation of replication and primosome activity, implying that this interaction has a critical role in assembly and progression of the SV40 replisome.

Sterol 14alpha-demethylase as a potential target for antitrypanosomal therapy: enzyme inhibition and parasite cell growth.

Sterol 14alpha-demethylases (CYP51) serve as primary targets for antifungal drugs, and specific inhibition of CYP51s in protozoan parasites Trypanosoma brucei (TB) and Trypanosoma cruzi (TC) might provide an effective treatment strategy for human trypanosomiases. Primary inhibitor selection is based initially on the cytochrome P450 spectral response to ligand binding. Ligands that demonstrate strongest binding parameters were examined as inhibitors of reconstituted TB and TC CYP51 activity in vitro. Direct correlation between potency of the compounds as CYP51 inhibitors and their antiparasitic effect in TB and TC cells implies essential requirements for endogenous sterol production in both trypanosomes and suggests a lead structure with a defined region most promising for further modifications. The approach developed here can be used for further large-scale search for new CYP51 inhibitors.

Role of the p68 subunit of human DNA polymerase alpha-primase in simian virus 40 DNA replication.

DNA polymerase alpha-primase (pol-prim) is a heterotetramer with DNA polymerase and primase activities. The polymerase (p180) and primase (p48 and p58) subunits synthesize primers and extend them, but the function of the remaining subunit (p68) is poorly understood. Genetic studies in yeast suggested an essential role for the p68 ortholog in early S phase prior to the hydroxyurea-sensitive step, possibly a regulatory role in initiation of DNA replication, but found no evidence for an essential function of p68 later in S phase. To investigate whether the human p68 subunit has an essential role in DNA replication, we examined the ability of a purified trimeric human pol-prim lacking p68 to initiate simian virus 40 DNA replication in vitro and to synthesize and elongate primers on single-stranded DNA in the presence of T antigen and replication protein A (RPA). Both activities of trimeric pol-prim were defective, but activity was recovered upon addition of separately purified p68. Phosphorylation of p68 by cyclin A-dependent protein kinase also inhibited both activities of pol-prim. The data strongly suggest that the p68 subunit is required for priming activity of pol-prim in the presence of RPA and T antigen, both during initiation at the origin and during lagging strand replication.

Trypanosome alternative oxidase: from molecule to function.

Trypanosome alternative oxidase (TAO) is the cytochrome-independent terminal oxidase of the mitochondrial electron transport chain. TAO is a diiron protein that transfers electrons from ubiquinol to oxygen, reducing the oxygen to water. The mammalian bloodstream forms of Trypanosoma brucei depend solely on TAO for respiration. The inhibition of TAO by salicylhydroxamic acid (SHAM) or ascofuranone is trypanocidal. TAO is present at a reduced level in the procyclic form of T. brucei, where it is engaged in respiration and is also needed for developmental processes. Alternative oxidases similar to TAO have been found in a wide variety of organisms but not in mammals, thus rendering TAO an important chemotherapeutic target for African trypanosomiasis.

Novel inhibitors of the trypanosome alternative oxidase inhibit Trypanosoma brucei brucei growth and respiration.

African trypanosomiasis is a deadly disease for which few chemotherapeutic options are available. The causative agents, Trypanosoma brucei rhodesiense and T. b. gambiense, utilize a non-cytochrome, alternative oxidase (AOX) for their cellular respiration. The absence of this enzyme in mammalian cells makes it a logical target for therapeutic agents. We designed three novel compounds, ACB41, ACD15, and ACD16, and investigated their effects on trypanosome alternative oxidase (TAO) enzymatic activity, parasite respiration, and parasite growth in vitro. All three compounds contain a 2-hydroxybenzoic acid moiety, analogous to that present in SHAM, and a prenyl side chain similar to that found in ubiquinol. ACD15 and ACD16 are further differentiated by the presence of a solubility-enhancing carbohydrate moiety. Kinetic studies with purified TAO show that all three compounds competitively inhibit TAO, and two compounds, ACB41 and ACD15, have inhibition constants five- and three-fold more potent than SHAM, respectively. All three compounds inhibited the respiration and growth of continuously cultured T. b. brucei bloodstream cells in a dose-dependent manner. None of the compounds interfered with respiration of rat liver mitochondria, nor did they inhibit the growth of a continuously cultured mammalian cell line. Collectively, the results suggest we have identified a new class of compounds that are inhibitors of TAO, have trypanocidal properties in vitro, and warrant further investigation in vivo.

The application of dose-rate volume histograms and survival fractions to multicellular dosimetry.

The distribution of therapeutic radiopharmaceuticals in volumes smaller than those that can be fully resolved by the imaging system, such as by PET and SPECT scanners, is usually assumed to be homogeneous. The aim of this study was to investigate the implications of such an assumption at a scale that can be defined as multicellular for heterogeneous activity localizations of (32)P, (90)Y, and (131)I. Dose-rate distributions from heterogeneous radioactivity uptakes have been calculated in cubic volumes of 1, 3, and 4 mm using the in-house software package DOVE. These have been studied by the use of dose-rate volume histograms, and the influence of the heterogeneous dose distribution on the treatment outcome has been analyzed by the calculation of Integral Survival Fractions. The results showed that the effect of the heterogeneous localization of the compound can be overridden by the amount of radioactivity administered. However, significant variations in the survival probability distributions have been observed, depending on the amount of initial activity considered, the activity configuration, the radionuclide, and the time over which the energy was deposited. It has been shown, for example, that the ability of longer-range beta emitters, such as (32)P and (90)Y, to invalidate heterogeneous dose-rate distributions may be negated by the decay rate of the radioactivity.

RMDP: a dedicated package for 131I SPECT quantification, registration and patient-specific dosimetry.

The limitations of traditional targeted radionuclide therapy (TRT) dosimetry can be overcome by using voxel-based techniques. All dosimetry techniques are reliant on a sequence of quantitative emission and transmission data. The use of (131)I, for example, with NaI or mIBG, presents additional quantification challenges beyond those encountered in low-energy NM diagnostic imaging, including dead-time correction and additional photon scatter and penetration in the camera head. The Royal Marsden Dosimetry Package (RMDP) offers a complete package for the accurate processing and analysis of raw emission and transmission patient data. Quantitative SPECT reconstruction is possible using either FBP or OS-EM algorithms. Manual, marker- or voxel-based registration can be used to register images from different modalities and the sequence of SPECT studies required for 3-D dosimetry calculations. The 3-D patient-specific dosimetry routines, using either a beta-kernel or voxel S-factor, are included. Phase-fitting each voxel's activity series enables more robust maps to be generated in the presence of imaging noise, such as is encountered during late, low-count scans or when there is significant redistribution within the VOI between scans. Error analysis can be applied to each generated dose-map. Patients receiving (131)I-mIBG, (131)I-NaI, and (186)Re-HEDP therapies have been analyzed using RMDP. A Monte-Carlo package, developed specifically to address the problems of (131)I quantification by including full photon interactions in a hexagonal-hole collimator and the gamma camera crystal, has been included in the dosimetry package. It is hoped that the addition of this code will lead to improved (131)I image quantification and will contribute towards more accurate 3-D dosimetry.

Cell-based Assays to Identify Inhibitors of Viral Disease.

BACKGROUND: Antagonizing the production of infectious virus inside cells requires drugs that can cross the cell membrane without harming host cells. OBJECTIVE: It is therefore advantageous to establish intracellular potency of anti-viral drug candidates early in the drug-discovery pipeline. METHODS: To this end, cell-based assays are being developed and employed in high-throughput drug screening, ranging from assays that monitor replication of intact viruses to those that monitor activity of specific viral proteins. While numerous cell-based assays have been developed and investigated, rapid counter screens are also needed to define the specific viral targets of identified inhibitors and to eliminate nonspecific screening hits. RESULTS/CONCLUSIONS: Here, we describe the types of cell-based assays being used in antiviral drug screens and evaluate the equally important counter screens that are being employed to reach the full potential of cell-based high-throughput screening.

Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex.

We report that during activation of the simian virus 40 (SV40) pre-replication complex, SV40 T antigen (Tag) helicase actively loads replication protein A (RPA) on emerging single-stranded DNA (ssDNA). This novel loading process requires physical interaction of Tag origin DNA-binding domain (OBD) with the RPA high-affinity ssDNA-binding domains (RPA70AB). Heteronuclear NMR chemical shift mapping revealed that Tag-OBD binds to RPA70AB at a site distal from the ssDNA-binding sites and that RPA70AB, Tag-OBD, and an 8-nucleotide ssDNA form a stable ternary complex. Intact RPA and Tag also interact stably in the presence of an 8-mer, but Tag dissociates from the complex when RPA binds to longer oligonucleotides. Together, our results imply that an allosteric change in RPA quaternary structure completes the loading reaction. A mechanistic model is proposed in which the ternary complex is a key intermediate that directly couples origin DNA unwinding to RPA loading on emerging ssDNA.

The trypanosome alternative oxidase exists as a monomer in Trypanosoma brucei mitochondria.

The bloodstream forms of African trypanosomes solely depend on trypanosome alternative oxidase (TAO), for respiration. Similar to alternative oxidases (AOXs) found in plants and fungi, TAO is a membrane-bound diiron protein. Here, we investigated if TAO exists as a dimer like plant AOXs, or as a monomer like that of fungi. We have found that TAO forms a homo-dimer on a regular SDS-PAGE in the absence of any reducing agent and exists as a monomer under reducing condition. However, TAO does not form a dimer upon treatment of mitochondria with diamide. TAO was found as a higher molecular mass complex on a Blue-native gel after solubilization with digitonin. In the detergent soluble form, TAO activity was stimulated under reducing and inhibited under oxidizing condition. However, these conditions have no effect on the TAO activity in the mitochondria. Moreover, chemical cross-linking analysis revealed that TAO could not be cross-linked when present in the mitochondria. Together, it suggests that like certain other hydrophobic membrane proteins, TAO forms a dimer or oligomer when solubilized with detergents, and the TAO-dimer is SDS-resistant. However, it exists as a monomer in Trypanosoma brucei mitochondria.

Mutational analysis of simian virus 40 T-antigen primosome activities in viral DNA replication.

The recruitment of DNA polymerase alpha-primase (pol-prim) is a crucial step in the establishment of a functional replication complex in eukaryotic cells, but the mechanism of pol-prim loading and the composition of the eukaryotic primosome are poorly understood. In the model system for simian virus 40 (SV40) DNA replication in vitro, synthesis of RNA primers at the origin of replication requires only the viral tumor (T) antigen, replication protein A (RPA), pol-prim, and topoisomerase I. On RPA-coated single-stranded DNA (ssDNA), T antigen alone mediates priming by pol-prim, constituting a relatively simple primosome. T-antigen activities proposed to participate in its primosome function include DNA helicase and protein-protein interactions with RPA and pol-prim. To test the role of these activities of T antigen in mediating priming by pol-prim, three replication-defective T antigens with mutations in the ATPase or helicase domain have been characterized. All three mutant proteins interacted physically and functionally with RPA and pol-prim and bound ssDNA, and two of them displayed some helicase activity. However, only one of these, 5030, mediated primer synthesis and elongation by pol-prim on RPA-coated ssDNA. The results suggest that a novel activity, present in 5030 T antigen and absent in the other two mutants, is required for T-antigen primosome function.

Reconstitution of human DNA polymerase delta using recombinant baculoviruses: the p12 subunit potentiates DNA polymerizing activity of the four-subunit enzyme.

Eukaryotic DNA polymerase delta is thought to consist of three (budding yeast) or four subunits (fission yeast, mammals). Four human genes encoding polypeptides p125, p50, p66, and p12 have been assigned as subunits of DNA polymerase delta. However, rigorous purification of human or bovine DNA polymerase delta from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. To reconstitute an intact DNA polymerase delta, we have constructed recombinant baculoviruses encoding the p125, p50, p66, and p12 subunits. From insect cells infected with four baculoviruses, protein preparations containing the four polypeptides of expected sizes were isolated. The four-subunit DNA polymerase delta displayed a specific activity comparable with that of the human, bovine, and fission yeast proteins isolated from natural sources. Recombinant DNA polymerase delta efficiently replicated singly primed M13 DNA in the presence of replication protein A, proliferating cell nuclear antigen, and replication factor C and was active in the SV40 DNA replication system. A three-subunit subcomplex consisting of the p125, p50, and p66 subunits, but lacking the p12 subunit, was also isolated. The p125, p50, and p66 polypeptides formed a stable complex that displayed DNA polymerizing activity 15-fold lower than that of the four-subunit polymerase. p12, expressed and purified individually, stimulated the activity of the three-subunit complex 4-fold on poly(dA)-oligo(dT) template-primer but had no effect on the activity of the four-subunit enzyme. Therefore, the p12 subunit is required to reconstitute fully active recombinant human DNA polymerase delta.

Insights into the oligomeric states, conformational changes, and helicase activities of SV40 large tumor antigen.

The large T (LT) antigen encoded by SV40 virus is a multi-domain, multi-functional protein that can not only transform cells but can also function as an efficient molecular machine to unwind duplex DNA for DNA replication. Here we report our findings on the oligomeric forms, domain interactions, and ATPase and helicase activities of various LT constructs. For the LT constructs that hexamerize, only two oligomeric forms, hexameric and monomeric, were detected in the absence of ATP/ADP. However, the presence of ATP/ADP stabilizes LT in the hexameric form. The LT constructs lacking the N- and C-terminal domains, but still retaining hexamerization ability, have ATPase as well as helicase activities at a level comparable to the full-length LT, suggesting the importance of hexamerization for these activities. The domain structures and the possible interactions between different LT fragments were probed with limited protease (trypsin) digestion. Such protease digestion generated a distinct pattern in the presence and absence of ATP/ADP and Mg(2+). The most C-terminal fragment (residues 628-708, containing the host-range domain), which was thought to be completely unstructured, was somewhat trypsin-resistant despite the presence of multiple Arg and Lys, possibly due to a rather structured C terminus. Furthermore, the N- and C-terminal fragments cleaved by trypsin were associated with other parts of the molecule, suggesting the interdomain interactions for the fragments at both ends.

Hepatitis C virus NS3 and simian virus 40 T antigen helicases displace streptavidin from 5'-biotinylated oligonucleotides but not from 3'-biotinylated oligonucleotides: evidence for directional bias in translocation on single-stranded DNA.

Helicases are enzymes that use energy from nucleoside triphosphate hydrolysis to unwind double-stranded (ds) DNA, a process vital to virtually every phase of DNA metabolism. Helicases have been classified as either 5'-to-3' or 3'-to-5' on the basis of their ability to unwind duplex DNA adjacent to either a 5' or 3' single-stranded (ss) DNA overhang. However, there has been debate as to whether this substrate preference is indicative of unidirectional translocation on ssDNA. We developed an assay that monitors the ability of a helicase to displace streptavidin from biotinylated oligonucleotides [Morris, P. D., and Raney, K. D. (1999) Biochemistry 38, 5164-5171]. Two helicases identified as having 5'-to-3' polarity displaced streptavidin from the 3'-end of biotinylated oligonucleotides but not from the 5'-end. We performed similar experiments using the 3'-to-5' helicases from the hepatitis C virus (NS3) and SV40 virus (SV40 T antigen). NS3 and SV40 T antigen were able to displace streptavidin from a 5'-biotinylated oligonucleotide but not from a 3'-biotinylated oligonucleotide. NS3 and SV40 T antigen enhanced the spontaneous rate of dissociation of streptavidin from biotin 340-fold and 1700-fold, respectively. The ssDNA binding protein, gp32, did not enhance dissociation of streptavidin from either end of an oligonucleotide. For NS3, the rate of displacement was faster from a 5'-biotinylated 60mer than from a 5'-biotinylated 30mer. The strong directional bias in streptavidin displacement activity exhibited by each helicase is consistent with a directional bias in translocation on ssDNA. The dependence of the reaction with NS3 on the oligonucleotide length suggests that multiple NS3 monomers are necessary for optimal activity.

A dominant-negative mutant of human DNA helicase B blocks the onset of chromosomal DNA replication.

A cDNA encoding a human ortholog of mouse DNA helicase B, which may play a role in DNA replication, has been cloned and expressed as a recombinant protein. The predicted human DNA helicase B (HDHB) protein contains conserved helicase motifs (superfamily 1) that are strikingly similar to those of bacterial recD and T4 dda proteins. The HDHB gene is expressed at low levels in liver, spleen, kidney, and brain and at higher levels in testis and thymus. Purified recombinant HDHB hydrolyzed ATP and dATP in the presence of single-stranded DNA, displayed robust 5'-3' DNA helicase activity, and interacted physically and functionally with DNA polymerase alpha-primase. HDHB proteins with mutations in the Walker A or B motif lacked ATPase and helicase activity but retained the ability to interact with DNA polymerase alpha-primase, suggesting that the mutants might be dominant over endogenous HDHB in human cells. When purified HDHB protein was microinjected into the nucleus of cells in early G(1), the mutant proteins inhibited DNA synthesis, whereas the wild type protein had no effect. Injection of wild type or mutant protein into cells at G(1)/S did not prevent DNA synthesis. The results suggest that HDHB function is required for S phase entry.