Recombinant Protein Production and Insect Cell Culture and Process

A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production

Mechanistic basis of the Cu(OAc)2 catalyzed azide-ynamine (3 + 2) cycloaddition reaction

R.P.B. and G.A.B. thank GSK and the Engineering and Physical Sciences Research Council (EPSRC) for an industrial CASE studentship (EP/P51066X/1). G.A.B., F.P., and A.J.B.W. thank the Leverhulme Trust (RP-2020-380). A.T.S. and G.A.B. thank the Biotechnology and Biological Research Council (BBSRC) for its support (BB/V017586/1; BB/T000627/1). A.J.B.W. and M.J.A. thank the EPSRC for its support (EP/R025754/1). A.J.B.W. thanks the Leverhulme Trust for a Research Fellowship (RF-2022-014).The Cu-catalyzed azide–alkyne cycloaddition (CuAAC) reaction is used as a ligation tool throughout chemical and biological sciences. Despite the pervasiveness of CuAAC, there is a need to develop more efficient methods to form 1,4-triazole ligated products with low loadings of Cu. In this paper, we disclose a mechanistic model for the ynamine-azide (3 + 2) cycloadditions catalyzed by copper(II) acetate. Using multinuclear nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy, and high-performance liquid chromatography analyses, a dual catalytic cycle is identified. First, the formation of a diyne species via Glaser–Hay coupling of a terminal ynamine forms a Cu(I) species competent to catalyze an ynamine-azide (3 + 2) cycloaddition. Second, the benzimidazole unit of the ynamine structure has multiple roles: assisting C–H activation, Cu coordination, and the formation of a postreaction resting state Cu complex after completion of the (3 + 2) cycloaddition. Finally, reactivation of the Cu resting state complex is shown by the addition of isotopically labeled ynamine and azide substrates to form a labeled 1,4-triazole product. This work provides a mechanistic basis for the use of mixed valency binuclear catalytic Cu species in conjunction with Cu-coordinating alkynes to afford superior reactivity in CuAAC reactions. Additionally, these data show how the CuAAC reaction kinetics can be modulated by changes to the alkyne substrate, which then has a predictable effect on the reaction mechanism.Peer reviewe

Topology of the pore-region of a K+ channel revealed by the NMR-derived structures of scorpion toxins

AbstractThe architecture of the pore-region of a voltage-gated K+ channel, Kv1.3, was probed using four high affinity scorpion toxins as molecular calipers. We established the structural relatedness of these toxins by solving the structures of kaliotoxin and margatoxin and comparing them with the published structure of charybdotoxin; a homology model of noxiustoxin was then developed. Complementary mutagenesis of Kv1.3 and these toxins, combined with electrostatic compliance and thermodynamic mutant cycle analyses, allowed us to identify multiple toxin-challel interactions. Our analyses reveals the existence of a shallow vestibule at the external entrance to the pore. This vestibule is ∼28−32A˚wide at its outer margin, ∼28−34A˚wide at its base, and ∼4−8A˚deep. The pore is 9–14A˚wide at its external entrance and tapers to a width of 4–5A˚at a depth of ∼5−7A˚from the vestibule. This structural information should directly aid in developing topological models of the pores of related ion channels and facilitate therapeutic drug design

Mechanistic basis of the Cu(OAc)2 catalyzed azide-ynamine (3+2)cycloaddition reaction

The Cu-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is used as a ligation tool throughout the chemical and biological sciences. Despite the pervasiveness of the CuAAC, there is a need to develop more efficient methods to form 1,4-triazole ligated products with low loadings of Cu. In this manuscript, we disclose a mechanistic model for the ynamine-azide (3+2)cycloadditions catalyzed by copper(II) acetate. Using multinuclear NMR spectroscopy, EPR spectroscopy and HPLC analyses, a dual catalytic cycle is identified. First, the formation of a diyne species via Glaser-Hay coupling of a terminal ynamine forms a Cu(I) species competent to catalyze an ynamine-azide (3+2)cycloaddition. Second, the benzimidazole unit of the ynamine structure effects multiple roles: assisting C-H activation, Cu-coordination and the formation of a post-reaction resting state Cu complex after completion of the (3+2)cycloaddition. Finally, reactivation of the Cu resting state complex is shown by addition of isotopically labelled ynamine and azide substrates to form a labelled 1,4-triazole product. This work provides a mechanistic basis for the use of mixed valency binuclear catalytic Cu species in conjunction with Cu-coordinating alkynes to afford superior reactivity in CuAAC reactions. Additionally, these data show how the CuAAC reaction kinetics can be modulated by changes to the alkyne substrate, which then has a predictable impact on the reaction mechanism

Human Embryonic Stem Cells and Embryonal Carcinoma Cells Have Overlapping and Distinct Metabolic Signatures

While human embryonic stem cells (hESCs) and human embryonal carcinoma cells (hECCs) have been studied extensively at the levels of the genome, transcriptome, proteome and epigenome our knowledge of their corresponding metabolomes is limited. Here, we present the metabolic signatures of hESCs and hESCs obtained by untargeted gas chromatography coupled to mass spectrometry (GC-MS). Whilst some metabolites are common to both cell types, representing the self-renewal and house-keeping signatures, others were either higher (e.g., octadecenoic acid, glycerol-3-phosphate, 4-hydroxyproline) or lower (e.g., glutamic acid, mannitol, malic acid, GABA) in hESCs (H9) compared to hECCs (NTERA2), these represent cell type specific signatures. Further, our combined results of GC-MS and microarray based gene expression profiling of undifferentiated and OCT4-depleted hESCs are consistent with the Warburg effect which is increased glycolysis in embryonic cells and tumor cells in the presence of O2 while oxidative phosphorylation (OXPHOS) is impaired or even shut down. RNAi-based OCT4 knock down mediated differentiation resulted in the activation of the poised OXPHOS machinery by expressing missing key proteins such as NDUFC1, UQCRB and COX, increase in TCA cycle activity and decreased lactate metabolism. These results shed light on the metabolite layer of pluripotent stem cells and could potentially establish novel metabolic markers of self renewal and pluripotency

Upsilon (1S+2S+3S) production in d+Au and p+p collisions at sqrt(s_NN)=200 GeV and cold-nuclear matter effects

The three Upsilon states, Upsilon(1S+2S+3S), are measured in d+Au and p+p collisions at sqrt(s_NN)=200 GeV and rapidities 1.2<|y|<2.2 by the PHENIX experiment at the Relativistic Heavy-Ion Collider. Cross sections for the inclusive Upsilon(1S+2S+3S) production are obtained. The inclusive yields per binary collision for d+Au collisions relative to those in p+p collisions (R_dAu) are found to be 0.62 +/- 0.26 (stat) +/- 0.13 (syst) in the gold-going direction and 0.91 +/- 0.33 (stat) +/- 0.16 (syst) in the deuteron-going direction. The measured results are compared to a nuclear-shadowing model, EPS09 [JHEP 04, 065 (2009)], combined with a final-state breakup cross section, sigma_br, and compared to lower energy p+A results. We also compare the results to the PHENIX J/psi results [Phys. Rev. Lett. 107, 142301 (2011)]. The rapidity dependence of the observed Upsilon suppression is consistent with lower energy p+A measurements.Comment: 495 authors, 11 pages, 9 figures, 5 tables. Submitted to Phys. Rev. C. Plain text data tables for the points plotted in figures for this and previous PHENIX publications are (or will be) publicly available at http://www.phenix.bnl.gov/papers.htm

Double Spin Asymmetry of Electrons from Heavy Flavor Decays in p+p Collisions at sqrt(s)=200 GeV

We report on the first measurement of double-spin asymmetry, A_LL, of electrons from the decays of hadrons containing heavy flavor in longitudinally polarized p+p collisions at sqrt(s)=200 GeV for p_T= 0.5 to 3.0 GeV/c. The asymmetry was measured at mid-rapidity (|eta|<0.35) with the PHENIX detector at the Relativistic Heavy Ion Collider. The measured asymmetries are consistent with zero within the statistical errors. We obtained a constraint for the polarized gluon distribution in the proton of |Delta g/g(log{_10}x= -1.6^+0.5_-0.4, {mu}=m_T^c)|^2 < 0.033 (1 sigma), based on a leading-order perturbative-quantum-chromodynamics model, using the measured asymmetry.Comment: 385 authors, 17 pages, 15 figures, 5 tables. Submitted to Phys. Rev. D. Plain text data tables for the points plotted in figures for this and previous PHENIX publications are (or will be) publicly available at http://www.phenix.bnl.gov/papers.htm

Wireworm : biology and nonchemical management in potatoes in the Pacific Northwest

Wireworm is the common name for the larvae of click beetles (Coleoptera: Family Elateridae). The adults do little or no damage, although there are some anecdotal reports that they can damage certain crops (e.g., grapes and stone fruits) by feeding on flowers. However, larval wireworms are among the most destructive of soil insect pests. They are important pests of potatoes and other crops, including corn, cereals, and carrots. Less frequently attacked are melons, beet roots, and strawberry fruits. Wireworms can also damage germinating seeds, but transplants are generally less susceptible. Their importance as crop pests seems to be increasing. This publication reviews the wireworm literature and provides information on wireworm biology, monitoring, risk assessment, and nonchemical control options that can be integrated into a variety of production systems.Published December 2008. Reviewed October 2013. Please check for up-to-date information in the OSU Extension Service Catalog: http://extension.oregonstate.edu/catalo

Measurements of elliptic and triangular flow in high-multiplicity 3^{3}He++Au collisions at sNN=200\sqrt{s_{_{NN}}}=200 GeV

We present the first measurement of elliptic ($v_2$) and triangular ($v_3$) flow in high-multiplicity $^{3}$He$+$Au collisions at $\sqrt{s_{_{NN}}}=200$ GeV. Two-particle correlations, where the particles have a large separation in pseudorapidity, are compared in $^{3}$He$+$Au and in $p$$+$$p$ collisions and indicate that collective effects dominate the second and third Fourier components for the correlations observed in the $^{3}$He$+$Au system. The collective behavior is quantified in terms of elliptic $v_2$ and triangular $v_3$ anisotropy coefficients measured with respect to their corresponding event planes. The $v_2$ values are comparable to those previously measured in $d$$+$Au collisions at the same nucleon-nucleon center-of-mass energy. Comparison with various theoretical predictions are made, including to models where the hot spots created by the impact of the three $^{3}$He nucleons on the Au nucleus expand hydrodynamically to generate the triangular flow. The agreement of these models with data may indicate the formation of low-viscosity quark-gluon plasma even in these small collision systems.Comment: 630 authors, 9 pages, 4 figures, 2 tables. v2 is the version accepted for publication by Physical Review Letters. Plain text data tables for the points plotted in figures for this and previous PHENIX publications are (or will be) publicly available at http://www.phenix.bnl.gov/papers.htm