Expression, purification and crystallization of the (3R)-hydroxyacyl-ACP dehydratase HadAB complex from Mycobacterium tuberculosis

AbstractThe (3R)-hydroxyacyl-ACP dehydratase HadAB, involved in the biosynthetic pathway for mycolic acid (MA) of Mycobacterium tuberculosis, catalyzes the third step in the fatty acid (FA) elongation cycle, which is an ideal and actual target for anti-tubercular agent. Though HadAB is predicted to be a member of the hotdog superfamily, it shares no sequence identity with typical hotdog fold isoenzyme FabZ. To characterize the significance of HadAB from the perspective of structural biology, large amount of pure HadAB complex is required for biochemical characterization and crystallization. Here, we used a unique expression and purification method. HadA and HadB were cloned separately and co-expressed in Escherichia coli. After GST affinity chromatography, two steps of anion exchange chromatography and gel filtration, the purity of the protein as estimated by SDS–PAGE was >95%. Using hanging-drop vapor-diffusion method, crystals were obtained and diffracted X-rays to 1.75Å resolution. The crystal belongs to space group P41212, with unit-cell parameters a=b=82.0Å, c=139.8Å, α=β=γ=90.0°

Quantum SDP Solvers: Large Speed-Ups, Optimality, and Applications to Quantum Learning

We give two new quantum algorithms for solving semidefinite programs (SDPs) providing quantum speed-ups. We consider SDP instances with m constraint matrices, each of dimension n, rank at most r, and sparsity s. The first algorithm assumes an input model where one is given access to an oracle to the entries of the matrices at unit cost. We show that it has run time O~(s^2 (sqrt{m} epsilon^{-10} + sqrt{n} epsilon^{-12})), with epsilon the error of the solution. This gives an optimal dependence in terms of m, n and quadratic improvement over previous quantum algorithms (when m ~~ n). The second algorithm assumes a fully quantum input model in which the input matrices are given as quantum states. We show that its run time is O~(sqrt{m}+poly(r))*poly(log m,log n,B,epsilon^{-1}), with B an upper bound on the trace-norm of all input matrices. In particular the complexity depends only polylogarithmically in n and polynomially in r. We apply the second SDP solver to learn a good description of a quantum state with respect to a set of measurements: Given m measurements and a supply of copies of an unknown state rho with rank at most r, we show we can find in time sqrt{m}*poly(log m,log n,r,epsilon^{-1}) a description of the state as a quantum circuit preparing a density matrix which has the same expectation values as rho on the m measurements, up to error epsilon. The density matrix obtained is an approximation to the maximum entropy state consistent with the measurement data considered in Jaynes\u27 principle from statistical mechanics. As in previous work, we obtain our algorithm by "quantizing" classical SDP solvers based on the matrix multiplicative weight update method. One of our main technical contributions is a quantum Gibbs state sampler for low-rank Hamiltonians, given quantum states encoding these Hamiltonians, with a poly-logarithmic dependence on its dimension, which is based on ideas developed in quantum principal component analysis. We also develop a "fast" quantum OR lemma with a quadratic improvement in gate complexity over the construction of Harrow et al. [Harrow et al., 2017]. We believe both techniques might be of independent interest

Structure of a Cell Entry Defective Human Adenovirus Provides Insights into Precursor Proteins and Capsid Maturation : Cryo-EM structure of ts1 virion of an adenovirus

Funding Information: We would like to thank Dr. Francisco Asturias for his advice on electron microscopy experiments and Dr. J.C. Ducom for installing Scipion and cisTEM packages on the HPC cluster and computational support in general. This work was supported by the NIH grant R21 AI146644 to V.S.R. Publisher Copyright: © 2021 Elsevier LtdMaturation of adenoviruses is distinguished by proteolytic processing of several interior minor capsid proteins and core proteins by the adenoviral protease and subsequent reorganization of adenovirus core. We report the results derived from the icosahedrally averaged cryo-EM structure of a cell entry defective form of adenovirus, designated ts1, at a resolution of 3.7 Å as well as of the localized reconstructions of unique hexons and penton base. The virion structure revealed the structures and organization of precursors of minor capsid proteins, pIIIa, pVI and pVIII, which are closely associated with the hexons on the capsid interior. In addition to a well-ordered helical domain (a.a. 310–397) of pIIIa, highlights of the structure include the precursors of VIII display significantly different structures near the cleavage sites. Moreover, we traced residues 4–96 of the membrane lytic protein (pVI) that includes an amphipathic helix occluded deep in the hexon cavity suggesting the possibility of co-assembly of hexons with the precursors of VI. In addition, we observe a second copy of pVI ordered up to residue L40 in the peripentonal hexons and a few fragments of density corresponding to 2nd and 3rd copies of pVI in other hexons. However, we see no evidence of precursors of VII binding in the hexon cavity. These findings suggest the possibility that differently bound pVI molecules undergo processing at the N-terminal cleavage sites at varying efficiencies, subsequently creating competition between the cleaved and uncleaved forms of VI, followed by reorganization, processing, and release of VI molecules from the hexon cavities.Peer reviewe

Quantum SDP Solvers: Large Speed-ups, Optimality, and Applications to Quantum Learning

We give two quantum algorithms for solving semidefinite programs (SDPs) providing quantum speed-ups. We consider SDP instances with m constraint matrices, each of dimension n, rank at most r, and sparsity s. The first algorithm assumes access to an oracle to the matrices at unit cost. We show that it has run time Õ(s^2(√((mϵ)^(−10)) + √((nϵ)^(−12))), with ϵ the error of the solution. This gives an optimal dependence in terms of m, n and quadratic improvement over previous quantum algorithms when m ≈ n. The second algorithm assumes a fully quantum input model in which the matrices are given as quantum states. We show that its run time is Õ (√m + poly(r))⋅poly(log m,log n,B,ϵ^(−1)), with B an upper bound on the trace-norm of all input matrices. In particular the complexity depends only poly-logarithmically in n and polynomially in r. We apply the second SDP solver to learn a good description of a quantum state with respect to a set of measurements: Given m measurements and a supply of copies of an unknown state ρ with rank at most r, we show we can find in time √m⋅poly(log m,log n,r,ϵ^(−1)) a description of the state as a quantum circuit preparing a density matrix which has the same expectation values as ρ on the m measurements, up to error ϵ. The density matrix obtained is an approximation to the maximum entropy state consistent with the measurement data considered in Jaynes' principle from statistical mechanics. As in previous work, we obtain our algorithm by "quantizing" classical SDP solvers based on the matrix multiplicative weight method. One of our main technical contributions is a quantum Gibbs state sampler for low-rank Hamiltonians with a poly-logarithmic dependence on its dimension, which could be of independent interest

Fatal attraction of Caenorhabditis elegans to predatory fungi through 6-methyl-salicylic acid

Salicylic acid is a phenolic phytohormone which controls plant growth and development. A methyl ester (MSA) derivative thereof is volatile and involved in plant-insect or plant-plant communication. Here we show that the nematode-trapping fungus Duddingtonia flagrans uses a methyl-salicylic acid isomer, 6-MSA as morphogen for spatiotemporal control of trap formation and as chemoattractant to lure Caenorhabditis elegans into fungal colonies. 6-MSA is the product of a polyketide synthase and an intermediate in the biosynthesis of arthrosporols. The polyketide synthase (ArtA), produces 6-MSA in hyphal tips, and is uncoupled from other enzymes required for the conversion of 6-MSA to arthrosporols, which are produced in older hyphae. 6-MSA and arthrosporols both block trap formation. The presence of nematodes inhibits 6-MSA and arthrosporol biosyntheses and thereby enables trap formation. 6-MSA and arthrosporols are thus morphogens with some functions similar to quorum-sensing molecules. We show that 6-MSA is important in interkingdom communication between fungi and nematodes

Interferon-γ Regulates Cellular Metabolism and mRNA Translation to Potentiate Macrophage Activation

Interferon-γ (IFN-γ) primes macrophages for enhanced inflammatory activation by Toll-like receptors (TLRs) and microbial killing, but little is known about the regulation of cell metabolism or mRNA translation during priming. We found that IFN-γ regulates human macrophage metabolism and translation by targeting the kinases mTORC1 and MNK that both converge on the selective regulator of translation initiation eIF4E. Physiological downregulation of mTORC1 by IFN-γ was associated with autophagy and translational suppression of repressors of inflammation such as HES1. Genome-wide ribosome profiling in TLR2-stimulated macrophages revealed that IFN-γ selectively modulates the macrophage translatome to promote inflammation, further reprogram metabolic pathways, and modulate protein synthesis. These results add IFN-γ-mediated metabolic reprogramming and translational regulation as key components of classical inflammatory macrophage activation

Chandra Detection of Three X-ray Bright Quasars at z>5

We report Chandra detection of three UV bright radio quiet quasars at $z\gtrsim5$. We have collected a sufficient number of photons to extract an X-ray spectrum of each quasar to measure their basic X-ray properties, such as the X-ray flux, power law photon index ($\Gamma$), and optical-to-X-ray spectral slope ($\alpha_{\rm OX}$). J074749+115352 at $z=5.26$ is the X-ray brightest radio-quiet quasar at $z>5$. It may have a short timescale variation (on a timescale of $\sim3800\rm~s$ in the observer's frame, or $\sim600\rm~s$ in the rest frame) which is however largely embedded in the statistical noise. We extract phase folded spectra of this quasar. There are two distinguishable states: a "high soft" state with an average X-ray flux $\sim2.7$ times of the "low hard" state, and a significantly steeper X-ray spectral slope ($\Gamma=2.40_{-0.32}^{+0.33}$ vs $1.78_{-0.24}^{+0.25}$). We also compare the three quasars detected in this paper to other quasar samples. We find that J074749+115352, with a SMBH mass of $M_{\rm SMBH}\approx1.8\times10^9\rm~M_\odot$ and an Eddington ratio of $\lambda_{\rm Edd}\approx2.3$, is extraordinarily X-ray bright. It has an average $\alpha_{\rm OX}=-1.46\pm0.02$ and a 2-10 keV bolometric correction factor of $L_{\rm bol}/L_{\rm2-10keV}=42.4\pm5.8$, both significantly depart from some well defined scaling relations. We compare $\Gamma$ of the three quasars to other samples at different redshifts, and do not find any significant redshift evolution based on the limited sample of $z>5$ quasars with reliable measurements of the X-ray spectral properties.Comment: 12 pages, 5 figures, 2 tables, accepted for publication by Ap

Oxytocin Regulates Synaptic Transmission in the Sensory Cortices in a Developmentally Dynamic Manner

The development and stabilization of neuronal circuits are critical to proper brain function. Synapses are the building blocks of neural circuits. Here we examine the effects of the neuropeptide oxytocin on synaptic transmission in L2/3 pyramidal neurons of the barrel field of the primary somatosensory cortex (S1BF). We find that perfusion of oxytocin onto acute brain slices significantly increases the frequency of miniature excitatory postsynaptic currents (mEPSC) of S1BF L2/3 pyramidal neurons at P10 and P14, but reduces it at the later ages of P22 and P28; the transition occurs at around P18. Since oxytocin expression is itself regulated by sensory experience, we also examine whether the effects of oxytocin on excitatory synaptic transmission correlate with that of sensory experience. We find that, indeed, the effects of sensory experience and oxytocin on excitatory synaptic transmission of L2/3 pyramidal neurons both peak at around P14 and plateau around P18, suggesting that they regulate a specific form of synaptic plasticity in L2/3 pyramidal neurons, with a sensitive/critical period ending around P18. Consistently, oxytocin receptor (Oxtr) expression in glutamatergic neurons of the upper layers of the cerebral cortex peaks around P14. By P28, however, Oxtr expression becomes more prominent in GABAergic neurons, especially somatostatin (SST) neurons. At P28, oxytocin perfusion increases inhibitory synaptic transmission and reduces excitatory synaptic transmission, effects that result in a net reduction of neuronal excitation, in contrast to increased excitation at P14. Using oxytocin knockout mice and Oxtr conditional knockout mice, we show that loss-of-function of oxytocin affects baseline excitatory synaptic transmission, while Oxtr is required for oxytocin-induced changes in excitatory synaptic transmission, at both P14 and P28. Together, these results demonstrate that oxytocin has complex and dynamic functions in regulating synaptic transmission in cortical L2/3 pyramidal neurons. These findings add to existing knowledge of the function of oxytocin in regulating neural circuit development and plasticity

HO-1 Is Essential for Tetrahydroxystilbene Glucoside Mediated Mitochondrial Biogenesis and Anti-Inflammation Process in LPS-Treated RAW264.7 Macrophages

2,3,5,4′-Tetrahydroxystilbene-2-O-β-D-glucoside (TSG), an important monomer extracted from Polygonum multiflorum, can prevent a number of inflammation associated chronic diseases. However, the mechanism involved in TSG inducing anti-inflammatory role remains unclear. As an inducible antioxidant enzyme, Heme oxygenase-1 (HO-1), is crucial for protecting the mammalian cells against adverse stimuli. Here, we found that the TSG treatment strongly induces the expression of HO-1 in an NRF2-depended manner. Meanwhile, TSG increased the mitochondrial mass through upregulation of the mitochondrial biogenesis activators (PGC-1α, NRF1, and TFAM) as well as the mitochondrial complex IV. Furthermore, TSG attenuated Lipopolysaccharide (LPS) mediated RAW264.7 cells activation and secretion of proinflammatory cytokines, including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Zinc Protoporphyrin (ZnPP), a selective inhibitor of HO-1 activity, was able to attenuate TSG mediated mitochondrial biogenesis and anti-inflammatory process. Finally, we observed that LPS induced obvious mtDNA depletion and ATP deficiency, which indicated a severe damage of mitochondria. TSG restored the LPS induced mitochondrial dysfunction via activation of the mitochondrial biogenesis. ZnPP treatment markedly reversed the inhibitory effects of TSG on mitochondrial damage and oxidative stress in LPS stimulated macrophages. Taken together, these findings suggest that TSG enhances mitochondrial biogenesis and function mainly via activation the HO-1. TSG can be developed as a potential drug for treatment of inflammatory diseases