Beyond iron: non-classical biological functions of bacterial siderophores

Bacteria secrete small molecules known as siderophores to acquire iron from their surroundings. For over 60 years, investigations into the bioinorganic chemistry of these molecules, including fundamental coordination chemistry studies, have provided insight into the crucial role that siderophores play in bacterial iron homeostasis. The importance of understanding the fundamental chemistry underlying bacterial life has been highlighted evermore in recent years because of the emergence of antibiotic-resistant bacteria and the need to prevent the global rise of these superbugs. Increasing reports of siderophores functioning in capacities other than iron transport have appeared recently, but reports of “non-classical” siderophore functions have long paralleled those of iron transport. One particular non-classical function of these iron chelators, namely antibiotic activity, was documented before the role of siderophores in iron transport was established. In this Perspective, we present an exposition of past and current work into non-classical functions of siderophores and highlight the directions in which we anticipate that this research is headed. Examples include the ability of siderophores to function as zincophores, chalkophores, and metallophores for a variety of other metals, sequester heavy metal toxins, transport boron, act as signalling molecules, regulate oxidative stress, and provide antibacterial activity.National Institutes of Health (U.S.) (R21 A1101784

Fluorescent chemosensors for exploring zinc metalloneurochemistry and detecting mercury in aqueous solution

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2006.Vita.Includes bibliographical references.Chapter 1. An Introduction to Zinc Metalloneurochemistry and Zinc Detection in Biology. This chapter presents an overview of zinc neurophysiology and pathology, which provides motivation for the design of new tools and tactics for zinc detection in vivo. A historical account of biological zinc detection is also given, followed by a summary of recent progress in the development and use of fluorescent Zn(II) sensors for in vivo studies. A summary of project goals and thesis organization is also included. Chapter 2. Zinc Sensors Based on Monosubstituted Fluorescein Platforms I: Routes to Electronic Variation, Syntheses and Spectroscopic Characterization. In this work, a convergent synthetic approach for the assembly of fluorescent zinc sensors from aniline-derivatized ligands and a fluroescein carboxaldehyde platform is presented. These sensors are based on the previously reported ZP4 motif and incorporate a di(2-picolyl)amine moiety in the aniline-based ligand framework. The effects of electronic variation, achieved by halogenation of either the zinc-binding unit or the fluorophore platform, on the fluorescence properties and aniline nitrogen atom pKa values are considered.(cont.) Chapter 3. Spectroscopic Characterization and Biological Applications of Halogenated Zinpyr Sensors. In this chapter, the effects of fluorescein halogenation on the photophysical properties and protonation equilbria of symmetrical Zinpyr derivatives, which contain two di(2-picolyl)amine-based ligand appendages, are considered. These sensors have sub-nM affinity for Zn(II) and are selective for Zn(II) over biologically relevant alkali and alkaline earth metals. Fluorescein halogenation influences background fluorescence, dynamic range, tertiary amine pKa, and both excitation and emission wavelengths. Extensive biological work, including cytotoxicity assays and confocal imaging, are also presented. Studies in a number of cell lines, including neurons, show that ZP3 is a versatile Zn(II) imaging tool. Chapter 4. Zinc Sensors Based on Monosubstituted Fluorescein Platforms II: Modulation of Zinc Affinity and Biological Applications. To access ZP sensors with lower Zn(II) affinity, pyrrole moieties were incorporated into an aniline-based ligand unit to give sensors ZP9 and ZP10. The photophysical characterization and metal binding properties of these sensors are described.(cont.) The pyrrole-for-pyridyl substitution affords Zn(II) sensors with improved Zn(II) selectivity and sub-[M dissociation constants. Biological imaging studies revealed that asymmetrical ZP probes, including ZP4, are cell permeable and Zn(II) responsive in vivo. Both ZP4 and ZP9 detect endogenous Zn(II) in acute hippocampal slices from the adult rat. Chapter 5. The Zinspy Family of Fluorescent Zinc Sensors: Syntheses and Spectroscopic Investigations. Four fluorescent sensors designed for Zn(II) detection and which contain a fluorescein reporting group and a pyridyl-amine-thioether derivatized ligand moiety were prepared and their photophyiscal properties characterized. These "Zinspy" sensors are water soluble and generally display -1.4 to -4.5-fold fluorescence enhancement upon Zn(II) coordination, depending upon fluorescein halogenation and the number and nature of the Zn(II)-binding appendages. The Zinspy sensors exhibit improved selectivity and lower affinity for Zn(II) compared to the di(2-picolyl)amine-based Zinpyr family members. Chapter 6. Zinspy Sensors with Enhanced Dynamic Range: Imaging Zinc Uptake and Mobilization with a Low Affinity Probe.(cont.) This chapter describes the preparation and characterization of Zinspy sensors containing non-coordinating thiophene heterocycles in the metal-binding unit. These probes show improved dynamic range relative to thioether-containing ZS sensors, low M dissociation constants for Zn(II) and improved Zn(II) selectivity. Stopped-flow kinetics investigations indicate fast association rates and reversible Zn(II) coordination with kon > 1.8 x 106 M'-s- and koff > 3 s-1 at 25 C. ZS5 is cell permeable, Zn(II)-responsive in vivo and localizes to the mitochondria of certain cell types. ZS5 can detect Zn(II) released from neurons following nitrosative stress. Chapter 7. QZ1 and QZ2, Rapid Reversible Quinoline Derivatized Fluoresceins for Sensing Biological Zinc. Two fluorescein-based dyes derivatized with 8-aminoquinoline were prepared and their photophysical, thermodynamic and zinc-binding kinetic properties determined. Because of their low background fluorescence and highly emissive Zn(II) complexes, QZ1 and QZ2 display a large dynamic range, with -42- and -150-fold fluorescence enhancements upon Zn(II) coordination, respectively.(cont.) These sensors have micromolar dissociation constants for Zn(II), improved selectivity and bind Zn(II) rapidly and reversibly with kon values >106 M-'s-1 and koffvalues of ~150 s-1. Biological imaging studies with ZP3 and QZ2 show that binding affinity is an important parameter for metal ion detection in vivo. QZ1 and QZ2 also respond to two-photon excitation and two-photon microscopy was used to visualize Zn(II) with QZ2 in live HeLa cells. Chapter 8. A "Turn-On" Fluorescent Sensor for the Selective Detection of Mercuric Ion in Aqueous Media. This chapter describes the synthesis, photophysical characterization and metal-binding properties of mercury sensor 1 (MS1). This sensor is based on a fluorescein platform and has a thioether-rich metal-binding unit, which conveys high selectivity for Hg(II). To the best of our knowledge, MS1 was the first reversible fluorescent Hg(II) sensor to give fluorescence turn-on in water. MS1 can detect low ppb levels of Hg(II) in aqueous solution at neutral pH. Chapter 9. Selective Hg(II) Detection in Aqueous Solution with Thiol Derivatized Fluoresceins. The syntheses and photophysical properties of MS2 and MS3, two asymmetrically derivatized fluorescein-based dyes designed for Hg(II) detection, are described.(cont.) These sensors each contain a single pyridyl-amine-thiol metal-binding moiety, form 1:1 complexes with Hg(II) and exhibit selectivity for Hg(II) over other Group 12 metals, alkali and alkaline earth metals, and most divalent first-row transition metals. Both dyes display superior brightness ( x ) and fluorescence enhancement following Hg(II) coordination in aqueous solution. At neutral pH, electron transfer (PET) quenching of the free dye is enhanced, and the Hg(II)-induced turn-on also benefits from alleviation of this pathway. MS2 can detect ppb levels of Hg(II) in aqueous solution, demonstrating its ability to identify environmentally relevant concentrations of Hg(II). Chapter 10. MS4, A Seminaphthofluorescein-Based Chemosensor for the Ratiometric Detection of Hg(II). The synthesis and photophysical characterization of MS4, an aniline-derivatized seminaphthofluorescein-based dye that contains a pyridyl-amine-thioether ligand analogous to that employed in the Zinspy Zn(II) sensor family (Chapter 5) are reported. Sensor MS4 provides single-excitation, dual-emission ratiometric detection of Hg(II) in aqueous solution. An -4-fold ratiometric change (624/524) is observed upon introduction of Hg(II) to an aqueous chloride-containing solution of MS4 at pH 8.(cont.) In this milieu, MS4 shows selectivity for Hg(II) over a background of alkali and alkaline earth metals, a number of divalent first-row transition metals and its Groupl2 congeners Zn(II) and Cd(II). Chapter 11. Turn-On and Ratiometric Mercury Sensing in Water with a Seminaphthofluorescein-Based Probe. The synthesis and characterization of MS5 are presented in this chapter. This sensor incorporates the aniline-derivatized thioether ligand used in the preparation of MS1 and the seminaphthofluorescein platform described in the design of MS4. MS5 gives selective fluorescence turn-on for Hg(II) at pH > 7. At pH > 8, single-excitation dual-emission ratiometric Hg(II) detection is possible by comparison of the (624 / 524) ratio before and after Hg(II) coordination. Studies of the pH dependence suggest that the seminaphthofluorescein dianion is critical for generating the ratiometric response. X-ray crystallographic studies with a salicylaldehyde-based model complex are presented to help elucidate the nature of Hg(II) coordination to MS1 and MS5. MS5 can respond to Hg(II) added to natural water samples, which points to its potential utility in the field.(cont.) Appendix 1. Miscellenous Fluorescein-Based Ligands. This appendix details the preparation and, in some cases, characterization of potential fluorescein based sensors for either Zn(II) or Hg(II) detection that were not described in earlier chapters. Many of these compounds give fluorescence turn-off or no fluorescence change with analyte binding. Sensors ZP11 and MS6 are lower-affinity probes that give fluorescence turn-on for Zn(II) and Hg(II), respectively. Appendix 2. Theoretical Investigations of Fluorescein Derivatives. In this chapter, we present the results from DFT and TDDFT calculations on fluorescein and its derivatives. These studies include establishing protocols for fluorescein pKa determination and TDDFT analysis. The absorption spectra of the fluorescein dianion and monoanion were assigned and a detailed molecular orbital analysis for a fluorescein dianion analog was conducted. These studies indicate that oxygen atoms in the xanthenone moiety influence the amount of C1 character in the donor and acceptor molecular orbitals responsible for fluorescein absorption.by Elizabeth M. Nolan.Ph.D

Bright Fluorescent Chemosensor Platforms for Imaging Endogenous Pools of Neuronal Zinc

AbstractA series of new fluorescent Zinpyr (ZP) chemosensors based on the fluorescein platform have been prepared and evaluated for imaging neuronal Zn2+. A systematic synthetic survey of electronegative substitution patterns on a homologous ZP scaffold provides a basis for tuning the fluorescence responses of “off-on” photoinduced electron transfer (PET) probes by controlling fluorophore pKa values and attendant proton-induced interfering fluorescence of the metal-free (apo) probes at physiological pH. We further establish the value of these improved optical tools for interrogating the metalloneurochemistry of Zn2+; the novel ZP3 fluorophore images endogenous stores of Zn2+ in live hippocampal neurons and slices, including the first fluorescence detection of Zn2+ in isolated dentate gyrus cultures. Our findings reveal that careful control of fluorophore pKa can minimize proton-induced fluorescence of the apo probes and that electronegative substitution offers a general strategy for tuning PET chemosensors for cellular studies. In addition to providing improved optical tools for Zn2+ in the neurosciences, these results afford a rational starting point for creating superior fluorescent probes for biological applications

On the existence of dyons and dyonic black holes in Einstein-Yang-Mills theory

We study dyonic soliton and black hole solutions of the ${\mathfrak {su}}(2)$ Einstein-Yang-Mills equations in asymptotically anti-de Sitter space. We prove the existence of non-trivial dyonic soliton and black hole solutions in a neighbourhood of the trivial solution. For these solutions the magnetic gauge field function has no zeros and we conjecture that at least some of these non-trivial solutions will be stable. The global existence proof uses local existence results and a non-linear perturbation argument based on the (Banach space) implicit function theorem.Comment: 23 pages, 2 figures. Minor revisions; references adde

Zfhx3-mediated genetic ablation of the SCN abolishes light entrainable circadian activity while sparing food anticipatory activity.

Circadian rhythms persist in almost all organisms and are crucial for maintaining appropriate timing in physiology and behaviour. Here, we describe a mouse mutant where the central mammalian pacemaker, the suprachiasmatic nucleus (SCN), has been genetically ablated by conditional deletion of the transcription factor Zfhx3 in the developing hypothalamus. Mutants were arrhythmic over the light-dark cycle and in constant darkness. Moreover, rhythms of metabolic parameters were ablated in vivo although molecular oscillations in the liver maintained some rhythmicity. Despite disruptions to SCN cell identity and circuitry, mutants could still anticipate food availability, yet other zeitgebers - including social cues from cage-mates - were ineffective in restoring rhythmicity although activity levels in mutants were altered. This work highlights a critical role for Zfhx3 in the development of a functional SCN, while its genetic ablation further defines the contribution of SCN circuitry in orchestrating physiological and behavioral responses to environmental signals

A Novel Whole Yeast-Based Subunit Oral Vaccine Against Eimeria tenella in Chickens

Cheap, easy-to-produce oral vaccines are needed for control of coccidiosis in chickens to reduce the impact of this disease on welfare and economic performance. Saccharomyces cerevisiae yeast expressing three Eimeria tenella antigens were developed and delivered as heat-killed, freeze-dried whole yeast oral vaccines to chickens in four separate studies. After vaccination, E. tenella replication was reduced following low dose challenge (250 oocysts) in Hy-Line Brown layer chickens (p<0.01). Similarly, caecal lesion score was reduced in Hy-Line Brown layer chickens vaccinated using a mixture of S. cerevisiae expressing EtAMA1, EtIMP1 and EtMIC3 following pathogenic-level challenge (4,000 E. tenella oocysts; p<0.01). Mean body weight gain post-challenge with 15,000 E. tenella oocysts was significantly increased in vaccinated Cobb500 broiler chickens compared to mock-vaccinated controls (p<0.01). Thus, inactivated recombinant yeast vaccines offer cost-effective and scalable opportunities for control of coccidiosis, with relevance to broiler production and chickens reared in low-and middle-income countries (LMICs)

Toll-Like Receptor mRNA Expression Is Selectively Increased in the Colonic Mucosa of Two Animal Models Relevant to Irritable Bowel Syndrome

Background: Irritable bowel syndrome (IBS) is largely viewed as a stress-related disorder caused by aberrant brain-gut– immune communication and altered gastrointestinal (GI) homeostasis. Accumulating evidence demonstrates that stress modulates innate immune responses; however, very little is known on the immunological effects of stress on the GI tract. Toll-like receptors (TLRs) are critical pattern recognition molecules of the innate immune system. Activation of TLRs by bacterial and viral molecules leads to activation of NF-kB and an increase in inflammatory cytokine expression. It was our hypothesis that innate immune receptor expression may be changed in the gastrointestinal tract of animals with stressinduced IBS-like symptoms. Methodology/Principal Findings: In this study, our objective was to evaluate the TLR expression profile in the colonic mucosa of two rat strains that display colonic visceral hypersensivity; the stress-sensitive Wistar-Kyoto (WKY) rat and the maternally separated (MS) rat. Quantitative PCR of TLR2-10 mRNA in both the proximal and distal colonic mucosae was carried out in adulthood. Significant increases are seen in the mRNA levels of TLR3, 4 & 5 in both the distal and proximal colonic mucosa of MS rats compared with controls. No significant differences were noted for TLR 2, 7, 9 & 10 while TLR 6 could not be detected in any samples in both rat strains. The WKY strain have increased levels of mRNA expression of TLR3, 4, 5, 7, 8, 9 & 10 in both the distal and proximal colonic mucosa compared to the control Sprague-Dawley strain. No significant differences in expression were found for TLR2 while as before TLR6 could not be detected in all samples in both strains. Conclusions: These data suggest that both early life stress (MS) and a genetic predisposition (WKY) to stress affect the expression of key sentinels of the innate immune system which may have direct relevance for the molecular pathophysiology of IBS

Impaired cholecystokinin-induced gallbladder emptying incriminated in spontaneous “black” pigment gallstone formation in germfree Swiss Webster mice

“Black” pigment gallstones form in sterile gallbladder bile in the presence of excess bilirubin conjugates (“hyperbilirubinbilia”) from ineffective erythropoiesis, hemolysis, or induced enterohepatic cycling (EHC) of unconjugated bilirubin. Impaired gallbladder motility is a less well-studied risk factor. We evaluated the spontaneous occurrence of gallstones in adult germfree (GF) and conventionally housed specific pathogen-free (SPF) Swiss Webster (SW) mice. GF SW mice were more likely to have gallstones than SPF SW mice, with 75% and 23% prevalence, respectively. In GF SW mice, gallstones were observed predominately in heavier, older females. Gallbladders of GF SW mice were markedly enlarged, contained sterile black gallstones composed of calcium bilirubinate and <1% cholesterol, and had low-grade inflammation, edema, and epithelial hyperplasia. Hemograms were normal, but serum cholesterol was elevated in GF compared with SPF SW mice, and serum glucose levels were positively related to increasing age. Aged GF and SPF SW mice had deficits in gallbladder smooth muscle activity. In response to cholecystokinin (CCK), gallbladders of fasted GF SW mice showed impaired emptying (females: 29%; males: 1% emptying), whereas SPF SW females and males emptied 89% and 53% of volume, respectively. Bilirubin secretion rates of GF SW mice were not greater than SPF SW mice, repudiating an induced EHC. Gallstones likely developed in GF SW mice because of gallbladder hypomotility, enabled by features of GF physiology, including decreased intestinal CCK concentration and delayed intestinal transit, as well as an apparent genetic predisposition of the SW stock. GF SW mice may provide a valuable model to study gallbladder stasis as a cause of black pigment gallstones.National Institutes of Health (U.S.) (Training Grant T32-OD10978-26)National Institutes of Health (U.S.) (Training Grant P30-ES002109)Kinship Foundation. Searle Scholars Progra

Quantization of fermions on Kerr space-time

We study a quantum fermion field on a background nonextremal Kerr black hole. We discuss the definition of the standard black hole quantum states (Boulware, Unruh, and Hartle-Hawking), focussing particularly on the differences between fermionic and bosonic quantum field theory. Since all fermion modes (both particle and antiparticle) have positive norm, there is much greater flexibility in how quantum states are defined compared with the bosonic case. In particular, we are able to define a candidate Boulware-like state, empty at both past and future null infinity, and a candidate Hartle-Hawking-like equilibrium state, representing a thermal bath of fermions surrounding the black hole. Neither of these states have analogues for bosons on a nonextremal Kerr black hole and both have physically attractive regularity properties. We also define a number of other quantum states, numerically compute differences in expectation values of the fermion current and stress-energy tensor between two states, and discuss their physical properties

'To live and die [for] Dixie': Irish civilians and the Confederate States of America

Around 20,000 Irishmen served in the Confederate army in the Civil War. As a result, they left behind, in various Southern towns and cities, large numbers of friends, family, and community leaders. As with native-born Confederates, Irish civilian support was crucial to Irish participation in the Confederate military effort. Also, Irish civilians served in various supporting roles: in factories and hospitals, on railroads and diplomatic missions, and as boosters for the cause. They also, however, suffered in bombardments, sieges, and the blockade. Usually poorer than their native neighbours, they could not afford to become 'refugees' and move away from the centres of conflict. This essay, based on research from manuscript collections, contemporary newspapers, British Consular records, and Federal military records, will examine the role of Irish civilians in the Confederacy, and assess the role this activity had on their integration into Southern communities. It will also look at Irish civilians in the defeat of the Confederacy, particularly when they came under Union occupation. Initial research shows that Irish civilians were not as upset as other whites in the South about Union victory. They welcomed a return to normalcy, and often 'collaborated' with Union authorities. Also, Irish desertion rates in the Confederate army were particularly high, and I will attempt to gauge whether Irish civilians played a role in this. All of the research in this paper will thus be put in the context of the Drew Gilpin Faust/Gary Gallagher debate on the influence of the Confederate homefront on military performance. By studying the Irish civilian experience one can assess how strong the Confederate national experiment was. Was it a nation without a nationalism