Rapid Analysis of Listeria monocytogenes Cell Wall Teichoic Acid Carbohydrates by ESI-MS/MS

We report the application of electrospray ionization (ESI) mass spectrometry for compositional characterization of wall teichoic acids (WTA), a major component of Gram-positive bacterial cell walls. Tandem mass spectrometry (ESI-MS/MS) of purified and chemically hydrolyzed monomeric WTA components provided sufficient information to identify WTA monomers and their specific carbohydrate constituents. A lithium matrix was used for ionization of uncharged WTA monomers, and successfully applied to analyze the WTA molecules of four Listeria strains differing in carbohydrate substitution on a conserved polyribitol-phosphate backbone structure. Carbohydrate residues such as N-acetylglucosamine or rhamnose linked to the WTA could directly be identified by ESI-MS/MS, circumventing the need for quantitative analysis by gas chromatography. The presence of a terminal N-acetylglucosamine residue tethered to the ribitol was confirmed using fluorescently labeled wheat-germ agglutinin. In conclusion, the mass spectrometry method described here will greatly facilitate compositional analysis and characterization of teichoic acids and similar macromolecules from diverse bacterial species, and represents a significant advance in the identification of serovar-specific carbohydrates and sugar molecules on bacteria

Advantages of a Mechanistic Codon Substitution Model for Evolutionary Analysis of Protein-Coding Sequences

A mechanistic codon substitution model, in which each codon substitution rate is proportional to the product of a codon mutation rate and the average fixation probability depending on the type of amino acid replacement, has advantages over nucleotide, amino acid, and empirical codon substitution models in evolutionary analysis of protein-coding sequences. It can approximate a wide range of codon substitution processes. If no selection pressure on amino acids is taken into account, it will become equivalent to a nucleotide substitution model. If mutation rates are assumed not to depend on the codon type, then it will become essentially equivalent to an amino acid substitution model. Mutation at the nucleotide level and selection at the amino acid level can be separately evaluated.The present scheme for single nucleotide mutations is equivalent to the general time-reversible model, but multiple nucleotide changes in infinitesimal time are allowed. Selective constraints on the respective types of amino acid replacements are tailored to each gene in a linear function of a given estimate of selective constraints. Their good estimates are those calculated by maximizing the respective likelihoods of empirical amino acid or codon substitution frequency matrices. Akaike and Bayesian information criteria indicate that the present model performs far better than the other substitution models for all five phylogenetic trees of highly-divergent to highly-homologous sequences of chloroplast, mitochondrial, and nuclear genes. It is also shown that multiple nucleotide changes in infinitesimal time are significant in long branches, although they may be caused by compensatory substitutions or other mechanisms. The variation of selective constraint over sites fits the datasets significantly better than variable mutation rates, except for 10 slow-evolving nuclear genes of 10 mammals. An critical finding for phylogenetic analysis is that assuming variable mutation rates over sites lead to the overestimation of branch lengths

Measurement of the Bottom-Strange Meson Mixing Phase in the Full CDF Data Set

We report a measurement of the bottom-strange meson mixing phase \beta_s using the time evolution of B0_s -> J/\psi (->\mu+\mu-) \phi (-> K+ K-) decays in which the quark-flavor content of the bottom-strange meson is identified at production. This measurement uses the full data set of proton-antiproton collisions at sqrt(s)= 1.96 TeV collected by the Collider Detector experiment at the Fermilab Tevatron, corresponding to 9.6 fb-1 of integrated luminosity. We report confidence regions in the two-dimensional space of \beta_s and the B0_s decay-width difference \Delta\Gamma_s, and measure \beta_s in [-\pi/2, -1.51] U [-0.06, 0.30] U [1.26, \pi/2] at the 68% confidence level, in agreement with the standard model expectation. Assuming the standard model value of \beta_s, we also determine \Delta\Gamma_s = 0.068 +- 0.026 (stat) +- 0.009 (syst) ps-1 and the mean B0_s lifetime, \tau_s = 1.528 +- 0.019 (stat) +- 0.009 (syst) ps, which are consistent and competitive with determinations by other experiments.Comment: 8 pages, 2 figures, Phys. Rev. Lett 109, 171802 (2012

Structure-Based Development of Small Molecule PFKFB3 Inhibitors: A Framework for Potential Cancer Therapeutic Agents Targeting the Warburg Effect

Cancer cells adopt glycolysis as the major source of metabolic energy production for fast cell growth. The HIF-1-induced PFKFB3 plays a key role in this adaptation by elevating the concentration of Fru-2,6-BP, the most potent glycolysis stimulator. As this metabolic conversion has been suggested to be a hallmark of cancer, PFKFB3 has emerged as a novel target for cancer chemotherapy. Here, we report that a small molecular inhibitor, N4A, was identified as an initial lead compound for PFKFB3 inhibitor with therapeutic potential. In an attempt to improve its potency, we determined the crystal structure of the PFKFB3•N4A complex to 2.4 Å resolution and, exploiting the resulting molecular information, attained the more potent YN1. When tested on cultured cancer cells, both N4A and YN1 inhibited PFKFB3, suppressing the Fru-2,6-BP level, which in turn suppressed glycolysis and, ultimately, led to cell death. This study validates PFKFB3 as a target for new cancer therapies and provides a framework for future development efforts

Hagfish and lamprey Hox genes reveal conservation of temporal colinearity in vertebrates

Hox genes exert fundamental roles for proper regional specification along the main rostro-caudal axis of animal embryos. They are generally expressed in restricted spatial domains according to their position in the cluster (spatial colinearity)—a feature that is conserved across bilaterians. In jawed vertebrates (gnathostomes), the position in the cluster also determines the onset of expression of Hox genes (a feature known as whole-cluster temporal colinearity (WTC)), while in invertebrates this phenomenon is displayed as a subcluster-level temporal colinearity. However, little is known about the expression profile of Hox genes in jawless vertebrates (cyclostomes); therefore, the evolutionary origin of WTC, as seen in gnathostomes, remains a mystery. Here, we show that Hox genes in cyclostomes are expressed according to WTC during development. We investigated the Hox repertoire and Hox gene expression profiles in three different species—a hagfish, a lamprey and a shark—encompassing the two major groups of vertebrates, and found that these are expressed following a whole-cluster, temporally staggered pattern, indicating that WTC has been conserved during the past 500 million years despite drastically different genome evolution and morphological outputs between jawless and jawed vertebrates

Role of 4-1BB Receptor in the Control Played by CD8+ T Cells on IFN-γ Production by Mycobacterium tuberculosis Antigen-Specific CD4+ T Cells

BACKGROUND: Antigen-specific IFN-gamma producing CD4(+) T cells are the main mediators of protection against Mycobacterium tuberculosis infection both under natural conditions and following vaccination. However these cells are responsible for lung damage and poor vaccine efficacy when not tightly controlled. Discovering new tools to control nonprotective antigen-specific IFN-gamma production without affecting protective IFN-gamma is a challenge in tuberculosis research. METHODS AND FINDINGS: Immunization with DNA encoding Ag85B, a candidate vaccine antigen of Mycobacterium tuberculosis, elicited in mice a low but protective CD4(+) T cell-mediated IFN-gamma response, while in mice primed with DNA and boosted with Ag85B protein a massive increase in IFN-gamma response was associated with loss of protection. Both protective and non-protective Ag85B-immunization generated antigen-specific CD8(+) T cells which suppressed IFN-gamma-secreting CD4(+) T cells. However, ex vivo ligation of 4-1BB, a member of TNF-receptor super-family, reduced the massive, non-protective IFN-gamma responses by CD4(+) T cells in protein-boosted mice without affecting the low protective IFN-gamma-secretion in mice immunized with DNA. This selective inhibition was due to the induction of 4-1BB exclusively on CD8(+) T cells of DNA-primed and protein-boosted mice following Ag85B protein stimulation. The 4-1BB-mediated IFN-gamma inhibition did not require soluble IL-10, TGF-beta, XCL-1 and MIP-1beta. In vivo Ag85B stimulation induced 4-1BB expression on CD8(+) T cells and in vivo 4-1BB ligation reduced the activation, IFN-gamma production and expansion of Ag85B-specific CD4(+) T cells of DNA-primed and protein-boosted mice. CONCLUSION/SIGNIFICANCE: Antigen-specific suppressor CD8(+) T cells are elicited through immunization with the mycobacterial antigen Ag85B. Ligation of 4-1BB receptor further enhanced their suppressive activity on IFN-gamma-secreting CD4(+) T cells. The selective expression of 4-1BB only on CD8(+) T cells in mice developing a massive, non-protective IFN-gamma response opens novel strategies for intervention in tuberculosis pathology and vaccination through T-cell co-stimulatory-based molecular targeting