MOLECULAR AND GENOMIC BASED INSIGHTS INTO THE EVOLUTION OF ENTEROCOCCUS FAECIUM FROM COMMENSAL TO HOSPITAL-ADAPTED PATHOGEN

The basis for the recent transition of Enterococcus faecium from a primarily commensal organism to one of the leading causes of hospital-acquired infections in the United States is not yet understood. To address this, the first part of my project assessed isolates from early outbreaks in the USA and South America using sequence analysis, colony hybridizations, and minimal inhibitory concentrations (MICs) which showed clinical isolates possess virulence and antibiotic resistance determinants that are less abundant or lacking in community isolates. I also revealed that the level of ampicillin resistance increased over time in clinical strains. By sequencing the pbp5 gene, I demonstrated an ~5% difference in the pbp5 gene between strains with MICs <4ug/ml and those with MICs >4µg/ml, but no specific sequence changes correlated with increases in MICs within the latter group. A 3-10% nucleotide difference was also seen in three other genes analyzed, which suggested the existence of two distinct subpopulations of E. faecium. This led to the second part of my project analyzing concatenated core gene sequences, SNPs, the 16S rRNA, and phylogenetics of 21 E. faecium genomes confirming two distinct clades; a community-associated (CA) clade and hospital-associated (HA) clade. Molecular clock calculations indicate that these two clades likely diverged ~ 300,000 to > 1 million years ago, long before the modern antibiotic era. Genomic analysis also showed that, in addition to core genomic differences, HA E. faecium harbor specific accessory genetic elements that may confer selection advantages over CA E. faecium. The third part of my project discovered 6 E. faecium genes with the newly identified “WxL” domain. My analyses, using RT-PCR, western blots, patient sera, whole-cell ELISA, and immunogold electron microscopy, indicated that E. faecium WxL genes exist in operons, encode bacterial cell surface localized proteins, that WxL proteins are antigenic in humans, and are more exposed on the surface of clinical isolates versus community isolates (even though they are ubiquitous in both clades). ELISAs and BIAcore analyses also showed that proteins encoded by these operons bind several different host extracellular matrix proteins, as well as to each other, suggesting a novel cell-surface complex. In summary, my studies provide new insights into the evolution of E. faecium by showing that there are two distantly related clades; one being more successful in the hospital setting. My studies also identified operons encoding WxL proteins whose characteristics could also contribute to colonization and virulence within this species.

Azotobacter vinelandii nitrogenase: “Kinetics of nif gene expression and insights into the roles of FdxN and NifQ in FeMo-co biosynthesis”

The Molybdenum-nitrogenase is responsible for most biological nitrogen fixation activity (BNF) in the biosphere. Due to its great agronomical importance, it has been the subject of profound genetic and biochemical studies. The Mo nitrogenase carries at its active site a unique iron-molybdenum cofactor (FeMoco) that consists of an inorganic 7 Fe, 1 Mo, 1 C, 9 S core coordinated to the organic acid homocitrate. Biosynthesis of FeMo-co occurs outside nitrogenase through a complex and highly regulated pathway involving proteins acting as molecular scaffolds, metallocluster carriers or enzymes that provide substrates in appropriate chemical forms. Specific expression regulatory factors tightly control the accumulation levels of all these other components. Insertion of FeMo-co into a P-cluster containing apo-NifDK polypeptide results in nitrogenase reconstitution. Investigation of FeMo-co biosynthesis has uncovered new radical chemistry reactions and new roles for Fe-S clusters in biology.

New insights into the regulation of NADPH oxidase dependent reactive oxygen species signaling during the plant immune response

Las NADPH oxidasas de plantas, denominadas “respiratory burst oxidase homologues” (RBOHs), producen especies reactivas del oxígeno (ROS) que median un amplio rango de funciones. En la célula vegetal, el ajuste preciso de la producción de ROS aporta la especificidad de señal para generar una respuesta apropiada ante las amenazas ambientales. RbohD y RbohF, dos de los diez genes Rboh de Arabidopsis, son pleiotrópicos y median diversos procesos fisiológicos en respuesta a patógenos. El control espacio-temporal de la expresión de los genes RbohD y RbohF podría ser un aspecto crítico para determinar la multiplicidad de funciones de estas oxidasas. Por ello, generamos líneas transgénicas de Arabidopsis con fusiones de los promoters de RbohD y RbohF a los genes delatores de la B-glucuronidasa y la luciferasa. Estas líneas fueron empleadas para revelar el patrón de expresión diferencial de RbohD y RbohF durante la respuesta inmune de Arabidopsis a la bacteria patógena Pseudomonas syringae pv. tomato DC3000, el hongo necrótrofo Plectosphaerella cucumerina y en respuesta a señales relacionadas con la respuesta inmune. Nuestros experimentos revelan un patrón de expresión diferencial de los promotores de RbohD y RbohF durante el desarrollo de la planta y en la respuesta inmune de Arabidopsis. Además hemos puesto de manifiesto que existe una correlación entre el nivel de actividad de los promotores de RbohD y RbohF con la acumulación de ROS y el nivel de muerte celular en respuesta a patógenos. La expression de RbohD y RbohF también es modulada de manera diferencial en respuesta a patrones moleculares asociados a patógenos (PAMPs) y por ácido abscísico (ABA). Cabe destacar que, mediante una estrategia de intercambio de promotores, hemos revelado que la región promotora de RbohD, es necesaria para dirigir la producción de ROS en respuesta a P. cucumerina. Adicionalmente, la activación del promotor de RbohD en respuesta al aislado de P. cucumerina no adaptado a Arabidopsis 2127, nos llevó a realizar ensayos de susceptibilidad con el doble mutante rbohD rbohF que han revelado un papel desconocido de estas oxidasas en resistencia no-huesped. La interacción entre la señalización dependiente de las RBOHs y otros componentes de la respuesta inmune de plantas podría explicar también las distintas funciones que median estas oxidasas en relación con la respuesta inmune. Entre la gran cantidad de señales coordinadas con la actividad de las RBOHs, existen evidencias genéticas y farmacológicas que indican que las proteínas G heterotriméricas están implicadas en algunas de las rutas de señalización mediadas por ROS derivadas de los RBOHs en respuesta a señales ambientales. Por ello hemos estudiado la relación entre estas RBOH-NADPH oxidasas y AGB1, la subunidad β de las proteínas G heterotriméricas en la respuesta inmune de Arabidopsis. Análisis de epistasis indican que las proteínas G heterotriméricas están implicadas en distintas rutas de señalización en defensa mediadas por las RBOHs. Nuestros resultados ilustran la relación compleja entre la señalización mediada por las RBOHs y las proteínas G heterotriméricas, que varía en función de la interacción planta-patógeno analizada. Además, hemos explorado la posible asociación entre AGB1 con RBOHD y RBOHF en eventos tempranos de la respuesta immune. Cabe señalar que experimentos de coímmunoprecipitación apuntan a una posible asociación entre AGB1 y la kinasa citoplasmática reguladora de RBOHD, BIK1. Esto indica un posible mecanismo de control de la función de esta NADPH oxidase por AGB1. En conjunto, estos datos aportan nuevas perspectivas sobre cómo, a través del control transcripcional o mediante la interacción con las proteínas G heterotriméricas, las NADPH oxidases de plantas median la producción de ROS y la señalización por ROS en la respuesta inmune. Nuestro trabajo ejemplifica cómo la regulación diferencial de dos miembros de una familia multigénica, les permite realizar distintas funciones fisiológicas especializadas usando un mismo mecanismo enzimático. ABSTRACT The plant NADPH oxidases, termed respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) which mediate a wide range of functions. Fine tuning this ROS production provides the signaling specificity to the plant cell to produce the appropriate response to environmental threats. RbohD and RbohF, two of the ten Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes in response to pathogens. One aspect that may prove critical to determine the multiplicity of functions of RbohD and RbohF is the spatio-temporal control of their gene expression. Thus, we generated Arabidopsis transgenic lines with RbohD- and RbohF-promoter fusions to the β-glucuronidase and the luciferase reporter genes. These transgenics were employed to reveal RbohD and RbohF promoter activity during Arabidopsis immune response to the pathogenic bacterium Pseudomonas syringae pv tomato DC3000, the necrotrophic fungus Plectosphaerella cucumerina and in response to immunity-related cues. Our experiments revealed a differential expression pattern of RbohD and RbohF throughout plant development and during Arabidopsis immune response. Moreover, we observed a correlation between the level of RbohD and RbohF promoter activity, the accumulation of ROS and the amount of cell death in response to pathogens. RbohD and RbohF gene expression was also differentially modulated by pathogen associated molecular patterns and abscisic acid. Interestingly, a promoter-swap strategy revealed the requirement for the promoter region of RbohD to drive the production of ROS in response to P. cucumerina. Additionally, since the RbohD promoter was activated during Arabidopsis interaction with a non-adapted P. cucumerina isolate 2127, we performed susceptibility tests to this fungal isolate that uncovered a new role of these oxidases on non-host resistance. The interplay between RBOH-dependent signaling with other components of the plant immune response might also explain the different immunity-related functions mediated by these oxidases. Among the plethora of signals coordinated with RBOH activity, pharmacological and genetic evidence indicates that heterotrimeric G proteins are involved in some of the signaling pathways mediated by RBOH–derived ROS in response to environmental cues. Therefore, we analysed the interplay between these RBOH-NADPH oxidases and AGB1, the Arabidopsis β-subunit of heterotrimeric G proteins during Arabidopsis immune response. We carried out epistasis studies that allowed us to test the implication of AGB1 in different RBOH-mediated defense signaling pathways. Our results illustrate the complex relationship between RBOH and heterotrimeric G proteins signaling, that varies depending on the type of plant-pathogen interaction. Furthermore, we tested the potential association between AGB1 with RBOHD and RBOHF during early immunity. Interestingly, our co-immunoprecipitation experiments point towards an association of AGB1 and the RBOHD regulatory kinase BIK1, thus providing a putative mechanism in the control of the NADPH oxidase function by AGB1. Taken all together, these studies provide further insights into the role that transcriptional control or the interaction with heterotrimeric G-proteins have on RBOH-NADPH oxidase-dependent ROS production and signaling in immunity. Our work exemplifies how, through a differential regulation, two members of a multigenic family achieve specialized physiological functions using a common enzymatic mechanism.

Automated data analysis to rapidly derive and communicate ecological insights from satellite-tag data : A case study of reintroduced red kites

Acknowledgements We thank Andrew Spink (Noldus Information Technology) and the Blogging Birds team members Peter Kindness and Abdul Adeniyi for their valuable contributions to this paper. John Fryxell, Chris Thaxter and Arjun Amar provided valuable comments on an earlier version. The study was part of the Digital Conservation project of dot.rural, the University of Aberdeen’s Digital Economy Research Hub, funded by RCUK (grant reference EP/G066051/1).

Insights, attitudes, and perceptions about asthma and its treatment : a multinational survey of patients from Europe and Canada

Acknowledgments Editorial assistance was provided by Ken Kauffman and Patricia Abramo, Adelphi Communications, New York, NY. This assistance was funded by Merck & Co., Inc.

Targeted disruption of the mouse gene encoding steroidogenic acute regulatory protein provides insights into congenital lipoid adrenal hyperplasia

An essential component of regulated steroidogenesis is the translocation of cholesterol from the cytoplasm to the inner mitochondrial membrane where the cholesterol side-chain cleavage enzyme carries out the first committed step in steroidogenesis. Recent studies showed that a 30-kDa mitochondrial phosphoprotein, designated steroidogenic acute regulatory protein (StAR), is essential for this translocation. To allow us to explore the roles of StAR in a system amenable to experimental manipulation and to develop an animal model for the human disorder lipoid congenital adrenal hyperplasia (lipoid CAH), we used targeted gene disruption to produce StAR knockout mice. These StAR knockout mice were indistinguishable initially from wild-type littermates, except that males and females had female external genitalia. After birth, they failed to grow normally and died from adrenocortical insufficiency. Hormone assays confirmed severe defects in adrenal steroids—with loss of negative feedback regulation at hypothalamic–pituitary levels—whereas hormones constituting the gonadal axis did not differ significantly from levels in wild-type littermates. Histologically, the adrenal cortex of StAR knockout mice contained florid lipid deposits, with lesser deposits in the steroidogenic compartment of the testis and none in the ovary. The sex-specific differences in gonadal involvement support a two-stage model of the pathogenesis of StAR deficiency, with trophic hormone stimulation inducing progressive accumulation of lipids within the steroidogenic cells and ultimately causing their death. These StAR knockout mice provide a useful model system in which to determine the mechanisms of StAR’s essential roles in adrenocortical and gonadal steroidogenesis.

Electrostatic steering and ionic tethering in enzyme–ligand binding: Insights from simulations

To bind at an enzyme’s active site, a ligand must diffuse or be transported to the enzyme’s surface, and, if the binding site is buried, the ligand must diffuse through the protein to reach it. Although the driving force for ligand binding is often ascribed to the hydrophobic effect, electrostatic interactions also influence the binding process of both charged and nonpolar ligands. First, electrostatic steering of charged substrates into enzyme active sites is discussed. This is of particular relevance for diffusion-influenced enzymes. By comparing the results of Brownian dynamics simulations and electrostatic potential similarity analysis for triose-phosphate isomerases, superoxide dismutases, and β-lactamases from different species, we identify the conserved features responsible for the electrostatic substrate-steering fields. The conserved potentials are localized at the active sites and are the primary determinants of the bimolecular association rates. Then we focus on a more subtle effect, which we will refer to as “ionic tethering.” We explore, by means of molecular and Brownian dynamics simulations and electrostatic continuum calculations, how salt links can act as tethers between structural elements of an enzyme that undergo conformational change upon substrate binding, and thereby regulate or modulate substrate binding. This is illustrated for the lipase and cytochrome P450 enzymes. Ionic tethering can provide a control mechanism for substrate binding that is sensitive to the electrostatic properties of the enzyme’s surroundings even when the substrate is nonpolar.

Ornaments of the earliest Upper Paleolithic: New insights from the Levant

Two sites located on the northern Levantine coast, Üçağızlı Cave (Turkey) and Ksar 'Akil (Lebanon) have yielded numerous marine shell beads in association with early Upper Paleolithic stone tools. Accelerator mass spectrometry (AMS) radiocarbon dates indicate ages between 39,000 and 41,000 radiocarbon years (roughly 41,000–43,000 calendar years) for the oldest ornament-bearing levels in Üçağızlı Cave. Based on stratigraphic evidence, the earliest shell beads from Ksar 'Akil may be even older. These artifacts provide some of the earliest evidence for traditions of personal ornament manufacture by Upper Paleolithic humans in western Asia, comparable in age to similar objects from Eastern Europe and Africa. The new data show that the initial appearance of Upper Paleolithic ornament technologies was essentially simultaneous on three continents. The early appearance and proliferation of ornament technologies appears to have been contingent on variable demographic or social conditions.

Insights into antibody catalysis: structure of an oxygenation catalyst at 1.9-angstrom resolution.

The x-ray crystal structures of the sulfide oxidase antibody 28B4 and of antibody 28B4 complexed with hapten have been solved at 2.2-angstrom and 1.9-angstrom resolution, respectively. To our knowledge, these structures are the highest resolution catalytic antibody structures to date and provide insight into the molecular mechanism of this antibody-catalyzed monooxygenation reaction. Specifically, the data suggest that entropic restriction plays a fundamental role in catalysis through the precise alignment of the thioether substrate and oxidant. The antibody active site also stabilizes developing charge on both sulfur and periodate in the transition state via cation-pi and electrostatic interactions, respectively. In addition to demonstrating that the active site of antibody 28B4 does indeed reflect the mechanistic information programmed in the aminophosphonic acid hapten, these high-resolution structures provide a basis for enhancing turnover rates through mutagenesis and improved hapten design.

Stabilizing and Modulating Color by Copigmentation: Insights from Theory and Experiment

Natural anthocyanin pigments/dyes and phenolic copigments/co-dyes form noncovalent complexes, which stabilize and modulate (in particular blue, violet, and red) colors in flowers, berries, and food products derived from them (including wines, jams, purees, and syrups). This noncovalent association and their electronic and optical implications constitute the copigmentation phenomenon. Over the past decade, experimental and theoretical studies have enabled a molecular understanding of copigmentation. This review revisits this phenomenon to provide a comprehensive description of the nature of binding (the dispersion and electrostatic components of π–π stacking, the hydrophobic effect, and possible hydrogen-bonding between pigment and copigment) and of spectral modifications occurring in copigmentation complexes, in which charge transfer plays an important role. Particular attention is paid to applications of copigmentation in food chemistry.

How young people from culturally and linguistically diverse backgrounds experience mental health: Some insights for mental health nurses

This article reports on part of a study that looked at the mental health of culturally and linguistically diverse (CALD) young people. The research sought to learn from CALD young people, carers, and service providers experiences relevant to the mental health of this group of young people. The ultimate goal was to gain insights that would inform government policy, service providers, ethnic communities and most importantly the young people themselves. To this end, qualitative interviews were undertaken with 123 CALD young people, 41 carers and 14 mental health service providers in Queensland, Western Australia and South Australia. Only one aspect of the study will be dealt with here, namely the views of the young CALD participants, which included risk factors, coping strategies and recommendations about how they could be supported in their struggle to maintain mental health. One of the most important findings of the study relates to the resilience of these young people and an insight into the strategies that they used to cope. The efforts of these young people to assist us in our attempts to understand their situation deserve to be rewarded by improvements in the care that we provide. To this end this article sets out to inform mental health nurses of the results of the study so that they will be in a position to better understand the needs and strengths of their CALD clients and be in a better position to work effectively with them.

Structural Insights into Viral Membrane Fusion Machinery via Cryo-Electron Tomography: Influenza Virus and Human Parainfluenza Virus

Thesis (Ph.D.)--University of Washington, 2016-06

New insights into NO-carbon and N2O-carbon reactions from quantum mechanical calculations

Density functional theory calculations were used to investigate the mechanisms of NO-carbon and N2O-carbon reactions. It was the first time that the importance of surface nitrogen groups was addressed in the kinetic behaviors of the NO-carbon reaction. It was found that the off-plane nitrogen groups that are adjacent to the zigzag edge sites and in-plane nitrogen groups that are located on the armchair sites make the bond energy of oxygen desorption even ca. 20% lower than that of the off-plane epoxy group adjacent to zigzag edge sites and in-plane o-quinone oxygen atoms on armchair sites; this may explain the reason why the experimentally obtained activation energy of the NO-carbon reaction is ca. 20% lower than that of the O-2-carbon reaction over 923 K. A higher ratio of oxygen atoms can be formed in the N2O-carbon reaction, because of the lower dissociation energy of N2O, which results in a higher ratio of off-plane epoxy oxygen atoms. The desorption energy of semiquinone with double adjacent off-plane oxygen groups is ca. 20% less than that of semiquinone with only one adjacent off-plane oxygen group. This may be the reason why the activation energy of N2O is also ca. 20% less than that of the O-2-carbon reaction. The new mechanism can also provide a good qualitative comparison for the relative reaction rates of NO-, N2O-, and O-2-carbon reactions. The anisotropic characters of these gas-carbon reactions can also be well explained.

The origin and evolution of the surfactant system in fish: Insights into the evolution of lungs and swim bladders

Several times throughout their radiation fish have evolved either lungs or swim bladders as gas-holding structures. Lungs and swim bladders have different ontogenetic origins and can be used either for buoyancy or as an accessory respiratory organ. Therefore, the presence of air-filled bladders or lungs in different groups of fishes is an example of convergent evolution. We propose that air breathing could not occur without the presence of a surfactant system and suggest that this system may have originated in epithelial cells lining the pharynx. Here we present new data on the surfactant system in swim bladders of three teleost fish ( the air-breathing pirarucu Arapaima gigas and tarpon Megalops cyprinoides and the non-air-breathing New Zealand snapper Pagrus auratus). We determined the presence of surfactant using biochemical, biophysical, and morphological analyses and determined homology using immunohistochemical analysis of the surfactant proteins (SPs). We relate the presence and structure of the surfactant system to those previously described in the swim bladders of another teleost, the goldfish, and those of the air-breathing organs of the other members of the Osteichthyes, the more primitive air-breathing Actinopterygii and the Sarcopterygii. Snapper and tarpon swim bladders are lined with squamous and cuboidal epithelial cells, respectively, containing membrane-bound lamellar bodies. Phosphatidylcholine dominates the phospholipid (PL) profile of lavage material from all fish analyzed to date. The presence of the characteristic surfactant lipids in pirarucu and tarpon, lamellar bodies in tarpon and snapper, SP-B in tarpon and pirarucu lavage, and SPs ( A, B, and D) in swim bladder tissue of the tarpon provide strong evidence that the surfactant system of teleosts is homologous with that of other fish and of tetrapods. This study is the first demonstration of the presence of SP-D in the air-breathing organs of nonmammalian species and SP-B in actinopterygian fishes. The extremely high cholesterol/disaturated PL and cholesterol/PL ratios of surfactant extracted from tarpon and pirarucu bladders and the poor surface activity of tarpon surfactant are characteristics of the surfactant system in other fishes. Despite the paraphyletic phylogeny of the Osteichthyes, their surfactant is uniform in composition and may represent the vertebrate protosurfactant.

New insights into ancient environments using dental characters in Australian Cenozoic lungfish

Environmentally-related wear conditions and pathologies affecting the dentition of fossil lungfish from freshwater deposits in Australia have been analysed and compared with similar changes in the dentition of the living Australian lungfish, Neoceratodus forsteri. Fossil populations from the Namba, Etadunna, Wipajiri and Katipiri formations in central Australia, and the Carl Creek Limestone and the Camfield beds in northern Australia were assessed. Tooth plates from populations of living lungfish from the Brisbane River and Enoggera Reservoir in southeast Queensland were analysed for comparison. Tooth plates were measured to determine the numbers of different age groups in each population. They were assessed for abrasion, attrition, spur and step wear, erosion and caries, and for trauma and pathological conditions such as malocclusion, hyperplasia, abscesses, osteopenia and parasitic damage. All of these conditions are related to the environment where the fish lived, are found in living members of the group, and can be compared directly with those of fossil relatives. The results suggest that some of the fossil populations were at risk before climatic changes late in the Cainozoic destroyed their habitats. Some fossil lungfish populations, such as those of the Wipajiri Formation, exhibit active spawning and recruitment, good growth rates and a low incidence of disease and environmentally related damage to the tooth plates. Others, like those of the Katipiri and Namba Formations, include no young, and the adult fish were ageing and show environmentally-related damage to the dentition. Etadunna lungfish had active recruitment, but the tooth plates show a high incidence of attrition and caries. Riversleigh lungfish were actively spawning but did not grow large. Tooth plates from this latter deposit have a high incidence of pathological conditions. Fish from the Camfield Beds, where food was severely limiting, had little serious pathology but high levels of caries. Pathologies among living lungfish are common, but fossil fish were comparatively healthy, with few serious dental problems. Information from studies of fossil lungfish confirms that conservation of the few living species of lungfish depends on the maintenance of clean environments that provide adequate supplies of food and suitable sites for spawning and for the growth of young fish.