Analysis of the NF-κB p50 dimer interface by diversity screening

An in vivo screen has been devised for NF-κB p50 activity in Escherichia coli exploiting the ability of the mammalian transcription factor to emulate a prokaryotic repressor. Active intracellular p50 was shown to repress the expression of a green fluorescent protein reporter gene allowing for visual screening of colonies expressing active p50 on agar plates. A library of mutants was constructed in which the residues Y267, L269, A308 and V310 of the dimer interface were simultaneously randomised and twenty-five novel functional interfaces were selected which repressed the reporter gene to similar levels as the wild-type protein. The leucine-269 alanine-308 core was repeatedly, but not exclusively, selected from the library whilst a diversity of predominantly non-polar residues were selected at positions 267 and 310. These results indicate that L269 and A308 may form a hot spot of interaction and allow an insight into the processes of dimer selectivity and evolution within this family of transcription factors.

Strategic targeting of the PI3K–NFκB axis in cisplatin-resistant NSCLC

Chemoresistance is a major therapeutic challenge to overcome in NSCLC, in order to improve the current survival rates of <15% at 5 years. We and others have shown increased PI3K signaling in NSCLC to be associated with a more aggressive disease, and a poorer prognosis. In this study, targeted inhibition of three strategic points of the PI3K–NFκB axis was performed with the aim of exploiting vulnerabilities in cisplatin-resistant NSCLC cells. Cisplatin-resistant cell lines were previously generated through prolonged exposure to the drug. Expression of PI3K and NFκB pathway-related genes were compared between cisplatin-resistant cells and their matched parent cells using a gene expression array, qRT-PCR, DNA sequencing, western blot, and immunofluorescence. Targeted inhibition was performed using GDC-0980, a dual PI3K–mTOR inhibitor currently in Phase II clinical trials in NSCLC, and DHMEQ, an inhibitor of NFκB translocation which has been used extensively both in vitro and in vivo. Effects of the two inhibitors were assessed by BrdU proliferation assay and multiparameter viability assay. NFKBIA was shown to be 12-fold overexpressed in cisplatin-resistant cells, with no mutations present in exons 3, 4, or 5 of the gene. Corresponding overexpression of IκBα was also observed. Treatment with DHMEQ (but not GDC-0980) led to significantly enhanced effects on viability and proliferation in cisplatin-resistant cells compared with parent cells. We conclude that NFκB inhibition represents a more promising strategy than PI3K–mTOR inhibition for treatment in the chemoresistance setting in NSCLC.

Synthesis, X-ray structure and catalytic properties of a copper(II) Schiff base complex modeling the activity of the Cu-B site of dopamine beta-hydroxylase

The copper(II) complex [Cu(salgly) (bpy)] . 4H(2)O (1), where salgly is a tridentate glycinatosalicylaldimine Schiffbase Ligand, is prepared and structurally characterized. The complex is found to be catalytically active in the oxidation of ascorbic acid by dioxygen and the process is also effective in the presence of benzylamine giving benzaldehyde as a product, thus modeling the activity of the Cu-B site of dopamine beta-hydroxylase. (C) 2000 Elsevier Science S.A. All rights reserved.

A Microscopic Theory of the f.c.c.-b.c.c. Interface .

We present a first-principles theory of the equilibrium b.c.c.-f.c.c. interface at coexistence using the density functional method. We assume that the interfacial region has local body-centred tetragonal (b.c.t.) symmetry and predict typical interfacial widths to be of order 2 to 3 lattice spacings with typical energies close to 0.05 J/m2. These quantities are in good agreement with laboratory measurements on coherent interfaces.

A subdivision-based implementation of the hierarchical b-spline finite element method

A novel technique is presented to facilitate the implementation of hierarchical b-splines and their interfacing with conventional finite element implementations. The discrete interpretation of the two-scale relation, as common in subdivision schemes, is used to establish algebraic relations between the basis functions and their coefficients on different levels of the hierarchical b-spline basis. The subdivision projection technique introduced allows us first to compute all element matrices and vectors using a fixed number of same-level basis functions. Their subsequent multiplication with subdivision matrices projects them, during the assembly stage, to the correct levels of the hierarchical b-spline basis. The proposed technique is applied to convergence studies of linear and geometrically nonlinear problems in one, two and three space dimensions. © 2012 Elsevier B.V.

Speciation and Phylogeography of Opsariichthys bidens (Pisces: Cypriniformes: Cyprinidae) in China: Analysis of the Cytochrome b Gene of mtDNA from Diverse Populations

Gao-Yan Li, Xu-Zhen Wang, Ya-Hui Zhao, Jie Zhang, Chun-Guang Zhang, and Shun-Ping He (2009) Speciation and phylogeography of Opsariichthys bidens (Pisces: Cypriniformes: Cyprinidae) in China: analysis of the cytochrome b gene of mtDNA from diverse populations. Zoological Studies 48(4): 569-583. The cyprinid fish Opsariichthys bidens Gunther is distributed in all major river systems of continental East Asia, and represents an attractive model for phylogeographic studies among cyprinid species or within a given species. In this study, we investigated the phylogeographic and demographic history of this species, using partial sequences of the cytochrome (cyt) b gene in mitochondrial (mt)DNA. Fish samples were collected from almost all major river systems where O. bidens is distributed in China. Sequence analysis showed remarkably high polymorphism, with 125 haplotypes in the 234 specimens examined, and with 89.8% of haplotypes occurring in only 1 specimen. A neutrality test indicated that some groups were not at mutation-drift equilibrium, suggesting a past population expansion. These results were supported by a mismatch distribution analysis. Based on our analysis, O. bidens consists of 4 groups belonging to 2 clades. The divergence time of the 2 clades was estimated to be 11.06-8.04 my. This value corresponds to the time of the 2nd uplift of the Qinghai-Tibet Plateau, the emergence of the East Asian monsoon, and the Epoch-6 Event. A two species scheme is proposed. http://zoolstud.sinica.edu.tw/Journals/48.4/569.pdf

Expression of the NF-κB inhibitor A20 is altered in the Cystic Fibrosis epithelium

Research Question: A20 is an LPS-inducible, cytoplasmic zinc finger protein, that inhibits TLR-activated NF-?B signalling by deubiquitinating TRAF6. A20 action is facilitated by complex formation with RNF11, Itch and TAX1BP1. This study investigates if the expression of A20 is altered in the chronically inflamed Cystic Fibrosis (CF) airway epithelium.<br/><br/>Methods: Nasal epithelial cells from CF patients (F508del homozygous), non-CF controls and immortalised epithelial cells (16HBE14o- and CFBE41o-) were stimulated with LPS. Cytoplasmic expression of A20 and expression of NF-?B subunits was analysed. Formation of the A20 ubiquitin editing complex was also investigated.<br/><br/>Results: In CFBE41o-, peak LPS-induced A20 expression was delayed compared with 16HBE14o- and fell significantly below basal levels 12-24 h after LPS stimulation. This was confirmed in primary CF airway cells. Additionally, a significant inverse relationship between A20 and p65 expression was observed. Inhibitor studies showed that A20 does not undergo proteasomal degradation in CFBE41o-. A20 interacted with TAX1BP1, RNF11 and TRAF6 in 16HBE14o- cells, but these interactions were not observed in CFBE41o-.<br/><br/>Conclusion: he expression of A20 is significantly altered in CF and important interactions with complex members and target proteins are lost, which may contribute to the state of chronic NF-?B-driven inflammation.

Recent changes to the biogeochemistry of Muchalat Inlet, B.C., and the implications for HABs

Senior thesis written for Oceanography 445

Reduced incorporation of the influenza B virus BM2 protein in virus particles decreases infectivity

BM2 is the fourth integral membrane protein encoded by the influenza B virus genome. It is synthesized late in infection and transported to the plasma membrane from where it is subsequently incorporated into progeny virus particles. It has recently been reported that BM2 has ion channel activity and may be the functional homologue of the influenza A virus M2 protein acting as an ion channel involved in viral entry. Using a reverse genetic approach it was not possible to recover virus which lacked BM2. A recombinant influenza B virus was generated in which the BM2 AUG initiation codon was mutated to GUG. This decreased the efficiency of translation of BM2 protein such that progeny virions contained only 1/8 the amount of BM2 seen in wild-type virus. The reduction in BM2 incorporation resulted in a reduction in infectivity although there was no concomitant decrease in the numbers of virions released from the infected cells. These data imply that the incorporation of sufficient BM2 protein into influenza B virions is required for infectivity of the virus particles. (C) 2004 Elsevier Inc. All rights reserved.

Identification of components of the Sigma B Regulon in Listeria monocytogenes that contribute to acid and salt tolerance

Sigma B (σB) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the ΔsigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The ΔsigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that σB contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the ΔsigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of σB in L. monocytogenes, we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the ΔsigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of σB. It also demonstrated clear roles for σB in both osmotic and low-pH stress tolerance and identified specific components of the σB regulon that contribute to the responses observed.

Analysis of the Hb(b)over-bar coupling constant and CP-violation effects at the future e(+)e(-) collider

The e(+)e(-)-->b (B) over bar nu(ν) over bar process, where nu is an electron, muon, or tau-lepton neutrino, is analyzed in detail for the general form of the coupling constant of a Higgs boson with b quarks, with the (m(b)/v)(a + igamma(5)b) parameterization of the Hb (b) over bar interaction. This process is shown to be highly sensitive to this coupling constant. Experiments at the future with roots = 500-GeV linear collider will provide limits of 2 and 20% for deviations of the parameters a and b, respectively, from their Standard Model values. Results concerning the e(+)e(-)-->b (b) over bar nu(ν) over bar process in combination with the independent measurements of the partial width Gamma(H --> b (b) over bar) can testify to the CP origin of the Higgs sector of the theory. (C) 2003 MAIK Nauka/Interperiodica.

Study of the decay B-s(0)->(DsDs(*))-D-(*)

We report a study of the decay B-s(0)->(DsDs(*))-D-(*) using a data sample corresponding to 1.3 fb(-1) of integrated luminosity collected by the D0 experiment in 2002-2006 during run II of the Fermilab Tevatron collider. One D-s((*)) meson was partially reconstructed in the decay D-s ->phi mu nu, and the other D-s((*)) meson was identified using the decay D-s ->phi pi where no attempt was made to distinguish D-s and D-s(*) states. For the branching fraction Br(B-s(0)->(DsDs(*))-D-(*)) we obtain a 90% C.L. range [0.002,0.080] and central value 0.039(-0.017)(+0.019)(stat)(-0.015)(+0.016)(syst). This was subsequently used to make the most precise estimate of the width difference Delta Gamma(CP)(s) in the B-s(0)-(B)over bar(s)(0) system: Delta Gamma(CP)(s)/Gamma(s)=0.079(-0.035)(+0.038)(stat)(-0.030)(+0.031)(syst).

Direct observation of the strange b baryon Xi(-)(b)

We report the first direct observation of the strange b baryon Xi(-)(b)(Xi) over bar (+)(b)). We reconstruct the decay Xi(-)(b)-->J/psi Xi(-), with J/psi-->mu(+)mu(-), and Xi(-)-->Lambda pi(-)-->p pi(-)pi(-) in p (p) over bar collisions at root s = 1.96 TeV. Using 1.3 fb(-1) of data collected by the D0 detector, we observe 15.2 +/- 4.4(stat)(-0.4)(+1.9)(syst) Xi(-)(b) candidates at a mass of 5.774 +/- 0.011(stat) +/- 0.015(syst) GeV. The significance of the observed signal is 5.5 sigma, equivalent to a probability of 3.3 x 10(-8) of it arising from a background fluctuation. Normalizing to the decay Lambda(b)-->J/psi Lambda, we measure the relative rate sigma(Xi(-)(b))xB(Xi(-)(b)-->J/psi Xi)/ sigma(Lambda(b))xB(Lambda(b)-->J/psi Lambda) = 0.28 +/- 0.09(stat)(-0.08)(+0.09)(syst).

Measurement of the Lambda(b) lifetime in the exclusive decay Lambda(b)-> J/psi Lambda

We have measured the Lambda(b) lifetime using the exclusive decay Lambda(b)-> J/psi Lambda, based on 1.2 fb(-1) of data collected with the D0 detector during 2002-2006. From 171 reconstructed Lambda(b) decays, where the J/psi and Lambda are identified via the decays J/psi ->mu(+)mu(-) and Lambda -> p pi, we measured the Lambda(b) lifetime to be tau(Lambda(b))=1.218(-0.115)(+0.130)(stat)+/- 0.042(syst) ps. We also measured the B-0 lifetime in the decay B-0 -> J/psi(mu(+)mu(-))K-S(0)(pi(+)pi(-)) to be tau(B-0)=1.501(-0.074)(+0.078)(stat)+/- 0.050(syst) ps, yielding a lifetime ratio of tau(Lambda(b))/tau(B-0)=0.811(-0.087)(+0.096)(stat)+/- 0.034(syst).

Measurement of the Lambda(b) cross section and the (Lambda)over-bar(b) to Lambda(b) ratio with J/psi Lambda decays in pp collisions at root s=7 TeV CMS Collaboration

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)