N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral Pathogen Tannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism

Tannerella forsythia is an anaerobic, Gram-negative oral pathogen that thrives in multispecies gingival biofilms associated with periodontitis. The bacterium is auxotrophic for the commonly essential bacterial cell wall sugar N-acetylmuramic acid (MurNAc) and, thus, strictly depends on an exogenous supply of MurNAc for growth and maintenance of cell morphology. A MurNAc transporter (Tf_MurT; Tanf_08375) and an ortholog of the Escherichia coli etherase MurQ (Tf_MurQ; Tanf_08385) converting MurNAc-6-phosphate to GlcNAc-6-phosphate were recently described for T. forsythia. In between the respective genes on the T. forsythia genome, a putative kinase gene is located. In this study, the putative kinase (Tf_MurK; Tanf_08380) was produced as a recombinant protein and biochemically characterized. Kinetic studies revealed Tf_MurK to be a 6-kinase with stringent substrate specificity for MurNAc exhibiting a 6 × 104 - fold higher catalytic efficiency (kcat/Km) for MurNAc than for N-acetylglucosamine (GlcNAc) with kcat values of 10.5 s−1 and 0.1 s−1 and Km values of 200 µM and 116 mM, respectively. The enzyme kinetic data suggest that Tf_MurK is subject to substrate inhibition (Ki[S] = 4.2 mM). To assess the role of Tf_MurK in the cell wall metabolism of T. forsythia, a kinase deletion mutant (1Tf_murK::erm) was constructed. This mutant accumulated MurNAc intracellularly in the exponential phase, indicating the capability to take up MurNAc, but inability to catabolize MurNAc. In the stationary phase, the MurNAc level was reduced in the mutant, while the level of the peptidoglycan precursor UDP-MurNAc-pentapeptide was highly elevated. Further, according to scanning electron microscopy evidence, the 1Tf_murK::erm mutant was more tolerant toward low MurNAc concentration in the medium (below 0.5 µg/ml) before transition from healthy, rod-shaped to fusiform cells occurred, while the parent strain required > 1 µg/ml MurNAc for optimal growth. These data reveal that T. forsythia readily catabolizes exogenous MurNAc but simultaneously channels a proportion of the sugar into peptidoglycan biosynthesis. Deletion of Tf_murK blocks MurNAc catabolism and allows the direction of MurNAc solely to peptidoglycan biosynthesis, resulting in a growth advantage in MurNAc-depleted medium. This work increases our understanding of the T. forsythia cell wall metabolism and may pave new routes for lead finding in the treatment of periodontitis

The IS History Initiative: Looking Forward by Looking Back

After officially appointing an AIS historian and forming the AIS history task force at the beginning of 2013, the AIS supported a set of systematic efforts, named IS history initiative, to preserve and represent the IS field’s history. From the perspective of the first AIS historian, I provide some background for the IS history initiative. Then I outline a detailed strategic plan and current status of its implementation. Ultimately, the IS history initiative has three goals: (1) to collect, represent, and preserve the IS field’s history; (2) to interpret, write, disseminate, and review the IS field’s history; and (3) to discover/identify IS genealogy, roots, sources, and facets that deserve to be examined from a historical point of view. Correspondingly, the strategic plan contains three parts. Each part has several specific tasks, many of which were already completed at the time of this writing, and several are either in progress or are planned for future efforts. This paper overviews both current efforts and guiding future efforts related to preserving and representing IS history

Electron-impact ionization cross sections of small molecules containing Fe and Cr *

J R acknowledges the PhD scholarship Grant PD/BD/142846/2018, and together with P L V, the Research Grants CEFITEC (UIDB/00068/2020) and PTDC/FISAQM/31281/2017 from the Portuguese National Funding Agency FCT. This work was also supported by the Radiation Biology and Biophysics Doctoral Training Programme (RaBBiT, PD/00193/2012); UCIBIO (UIDB/04378/2020). The work has partially been carried out within the framework of the EUROfusion Consortium and received funding from the Euratom research and training programme by Grant Agreement No. 101052200-EUROfusion. The views and opinions expressed herein do not necessarily reflect those of the European Commission. The computational results have been obtained using the HPC infrastructure LEO of the University of Innsbruck.We present the electron-impact ionization cross sections(EICSs) of iron and chromium hydrides, nitrides, and oxides. The motivation of this work stems from the fact that chemical sputtering from a steel surface exposed to a hot plasma can create these molecules which in turn influence the composition and energy balance of the plasma. The latter influence is quantified by the EICS which we derive by using two semi-empirical methods which can be employed in the relevant energy range of 10-1000 eV. They are important molecular properties for plasma- and materials science. We discuss the foundations of the methods and present the cross sections of the high- and low-spin states of the species in their neutral ground states and of their cations.publishersversionpublishe

Moderating and mediating roles of human capital and competitive advantage on entrepreneurial orientation, social network and performance of SMEs in Nigeria

The main objective of this study is to examine the mediating role of competitive advantage (CA) and the moderating role of human capital (HC) on the relationships between entrepreneurial orientation (EO), social network (SN), and performance of small and medium enterprises (SMEs) in Nigeria. Data were collected from the SMEs operating in North Western Nigeria using a quantitative survey design. The study adopted a systematic random sampling, selected respondents from the population of 8,286 SMEs in Kano State Nigeria, and questionnaires distributed through the postal method. A total of 283 usable questionnaires were returned, giving a response rate of 38.45%. Partial Least Squares-Structural Equation Modeling (PLS-SEM) was used to test the study hypotheses. The findings revealed that EO, SN, HC and CA are important strategic orientations for the performance of SMEs in Nigeria. The findings also revealed that SME performance depends on the degree of EO, HC and SN of the firm. It further shows that EO, SN and HC positively influence performance, and human capital moderates the relationship between EO and firm performance, but the findings indicate that HC does not moderate the relationship between SN and SME performance. However, competitive advantage was found to mediate the relationships between EO, SN, and performance. The findings of this study provide important insights to owner/managers of SMEs, policy makers and researchers to further understand the effects of EO, SN, HC and CA on SME performance. SMEs should also be encouraged to improve their EO, SN and HC which may increase their performances. Finally, the study implications, limitations as well suggestions for future research are discussed

Separación y caracterización de glicopéptidos y glicanos en fluidos biológicos. Aplicación al diagnóstico del cáncer y otras patologías

[spa] La presente tesis doctoral se centra en el desarrollo de nuevos métodos de análisis para la identificación y separación de glicopéptidos y glicanos utilizando técnicas microseparativas acopladas a la espectrometría de masas (cromatografía de líquidos capilar y electroforesis capilar, CapLC-MS y CE-MS). Aunque el análisis de los glicopéptidos es una estrategia habitualmente utilizada para la caracterización de productor farmacéuticos, continúa siendo difícil la detección de glicoformas poco abundantes que podrían ser esenciales para diferenciar proteínas endógenas de variantes recombinantes. De este modo, en esta tesis se han desarrollado dos métodos para la purificación selectiva de glicopéptidos, obtenidos de digestos de glicoproteínas, utilizando la eritropoetina humana recombinante (rhEPO) como glicoproteína modelo. Por otro lado, en muchas patologías se han descrito alteraciones en los glicanos de ciertas glicoproteínas en presencia de procesos inflamatorios importantes y muchos tipos de cáncer. Además, dado que ciertas estructuras de glicano así como tipos de enlace han estado relacionados con enfermedades específicas, la caracterización exhaustiva de los tipos de enlace de los isómeros de los glicanos ha suscitado gran interés biomédico en los últimos años. En este sentido, se han establecido en esta tesis doctoral diferentes metodologías para la caracterización de los isómeros de los glicanos de la alfa-1-glicoproteína ácida (hAGP), la cual ha generado interés biomédico dada la alteración de sus glicanos en ciertos procesos inflamatorios y cáncer. Primero, la digestión con diversas exoglicosidasas (sialidasa, fucosidasa y galactosidasa) en combinación con el análisis de los glicanos per cromatografía de líquidos capilar de interacción hidrofílica zwitteriónica acoplada a la espectrometría de masas (CapZIC-HILIC-MS) y la estrategia GRIL utilizando [12C6]/[13C6]- anilina, permitió asignar los enlaces de los ácidos siálicos, así como de las fucosas de los isómeros de los glicanos de la hAGP. Por otro lado, se desarrolló un método para asignar los isómeros de los glicanos per espectrometría de masas en tándem de alta resolución. La posibilidad de separar los isómeros antes de la detección, juntamente con la obtención de la masa exacta de los iones fragmentos, permitió el establecimiento de un método de caracterización de los glicanos fiable por CapZIC-HILIC-MS/MS. Además, la doctoranda realizó una estancia en la Freie Universität Berlin (Berlin, Alemania), bajo la supervisión del Dr. Kevin Pagel, para estudiar la separación de los isómeros de glicanos per espectrometría de movilidad iónica acoplada a la espectrometría de masas (IM-MS). Finalmente, el método por CapZIC-HILIC-MS en combinación con la estrategia GRIL utilizando [12C6]/[13C6]-anilina se aplicó en esta tesis para el análisis de muestras patológicas, con el fin de evaluar posibles alteraciones en los isómeros de los glicanos de dos glicoproteínas de fase aguda, l’hAGP y la transferrina de ratón (mTf), en presencia de ciertos procesos inflamatorios y cáncer. Además, se emplearon herramientas quimiométricas con el objetivo de identificar glicanos como candidatos biomarcadores de estas patologías.[eng] The present thesis is focused on the development of novel analytical methods for the identification and separation of glycopeptides and glycans by microseparative techniques coupled to mass spectrometry. Although the analysis of glycopeptides is a commonly used strategy for glycan characterization of biopharmaceuticals, the detection of low abundant glycopeptides that could be essential to differentiate endogenous and recombinant variants of certain proteins continues to be a challenge. In this thesis, methods to selectively enrich glycopeptides from glycoprotein digests were developed using recombinant human erythropoietin (rhEPO) as model glycoprotein. On the other hand, alterations in glycan structures have been described in presence of important inflammatory processes and many types of cancer. As certain glycan isomers or linkage-types have been related to specific pathologies, the in depth characterization of glycan isomer linkage-types has aroused great interest in last years. In this regard, different methodologies were established in this thesis to characterize human alpha-1-acid glycoprotein (hAGP) glycan isomers, whose glycosylation has been described to be altered in several diseases. Exoglycosidase digestions, tandem mass spectrometry (MS/MS) as well as ion-mobility mass spectrometry (IM-MS) were used with this purpose. Finally, the established method for glycan isomer separation in combination with the GRIL strategy with [12C6]/[13C6]aniline was used in this thesis to evaluate possible glycan isomer modifications in two acute-phase proteins, hAGP and mouse transferrin (mTf), caused by certain inflammatory diseases and cancer. Moreover, multivariate data tools, such as PCA and PLS-DA, were also employed in these studies to identify glycan-based biomarker candidates

Quantitative Bottom-Up and Top-Down LC/FT-ICR MS for Multisite Phosphoproteins and Natural Product Biosynthetic Pathways.

Bottom-up (involving proteolysis) and top-down (intact proteins) mass spectrometry (MS)-based proteomics are increasingly important tools for protein identification and quantitation from complex mixtures. A multitude of quantitation techniques are available, each with advantages and disadvantages, from label to label-free and relative to absolute quantitation. The ultra-high resolution and mass accuracy of Fourier transform ion cyclotron resonance (FT-ICR) MS, can improve current quantitation methods. This thesis presents FT-ICR MS-based quantification of proteins and peptides with dynamic post-translational modifications, and a novel data-independent liquid chromatography (LC)/FT-ICR MS approach for targeted discovery of novel natural product biosynthetic pathways. A novel negative ion mode quantitation method was developed involving standard peptides with Val to Leu or Leu to Val substitutions, resulting in similar ionization efficiencies. This method was applied to Sic1, a yeast protein with a switch-like response when six out of nine phosphorylation sites are phosphorylated. With this quantitation technique, the phosphorylation kinetics of all nine sites were determined. Relative FT-ICR MS-based quantitation was applied to marine natural product biosynthetic enzymes. Ketosynthase, acyl transferase, and acyl carrier protein (ACP) active site occupancies were identified and quantified via LC/FT-ICR MS, as PikAIII, module 5 in the pikromycin biosynthetic pathway, completed its catalytic cycle with the addition of pentaketide substrate, methyl-malonyl extender unit, and NADPH. Several protocol changes were made to observe the pentaketide bound to the ketosynthase active site for the first time. The largely homologous jamaicamide (Jam) and curacin (Cur) biosynthetic pathways diverge at an enoyl reductase (ER) domain. Catalytic efficiencies of wildtype and mutant ER domains were measured by quantifying ACP-bound product via top-down FT-ICR MS and collision-induced dissociation. Cyclopropanase activity of a novel chimeric ER, designed by inserting a Cur ER loop into Jam ER was measured via FT-ICR MS. Finally, a novel data-independent acquisition method was developed for targeted discovery of novel natural product biosynthetic pathways from unsequenced organisms. This technique utilizes the resonant IR absorptivity of a secondary phosphate in the ACP phosphopantetheine modification. Peptides with this modification dissociate with high efficiency upon IR irradiation, whereas unmodified peptides are largely unaffected by IR irradiation.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113355/1/wendhale_1.pd

Brave New GES World:A Systematic Literature Review of Gestures and Referents in Gesture Elicitation Studies

How to determine highly effective and intuitive gesture sets for interactive systems tailored to end users’ preferences? A substantial body of knowledge is available on this topic, among which gesture elicitation studies stand out distinctively. In these studies, end users are invited to propose gestures for specific referents, which are the functions to control for an interactive system. The vast majority of gesture elicitation studies conclude with a consensus gesture set identified following a process of consensus or agreement analysis. However, the information about specific gesture sets determined for specific applications is scattered across a wide landscape of disconnected scientific publications, which poses challenges to researchers and practitioners to effectively harness this body of knowledge. To address this challenge, we conducted a systematic literature review and examined a corpus of N=267 studies encompassing a total of 187, 265 gestures elicited from 6, 659 participants for 4, 106 referents. To understand similarities in users’ gesture preferences within this extensive dataset, we analyzed a sample of 2, 304 gestures extracted from the studies identified in our literature review. Our approach consisted of (i) identifying the context of use represented by end users, devices, platforms, and gesture sensing technology, (ii) categorizing the referents, (iii) classifying the gestures elicited for those referents, and (iv) cataloging the gestures based on their representation and implementation modalities. Drawing from the findings of this review, we propose guidelines for conducting future end-user gesture elicitation studies

Quantitative LC-HRMS determination of selected cardiovascular drugs, in dried blood spots, as an indicator of adherence to medication

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.Dried blood spot (DBS) sampling was investigated as a means of obtaining micro-volume blood samples for the quantitative analyses of ten commonly UK prescribed cardiovascular drugs as an indicator of medication adherence. An 8 mm disc was punched out from each DBS from calibration, quality control and volunteer samples and extracted using methanol containing the internal standard. Each extract was evaporated to dryness, the residue reconstituted in methanol:water (40:60 v/v) containing 0.1% formic acid and analysed by LC-HRMS. Chromatography was performed using gradient elution on a Zorbax Eclipse C18 HD 100 mmx2.1 mm, 1.8 µm pore size column with the column oven temperature at 40˚C. Flow rate of the mobile phase was 0.6ml/min with a run time of 2.5 min. Electrospray positive ionization was used for MS detection. Drug recoveries from spiked blood spots were 68% for simvastatin and ≥ 87% for all other target drugs. Compound specificity was obtained operating the MS with a 5ppm mass window. The LC-HRMS method was validated, with results for accuracy and precision within acceptable limits; analytes were stable at room temperature for at least 10 weeks and different blood spot volumes and haematocrit values had no significant effect. The LC-HRMS assay was used to analyse DBS samples from volunteers, some of whom were prescribed one or more of the target drugs. In results from 37 volunteers the assay successfully identified volunteers who were known to be either adherent or nonadherent; confirmed the correct drug/drugs for multiple prescriptions; demonstrated no false positives from other cardiovascular drugs; revealed several examples of unsuspected non-adherence. These results indicated that the developed assay was suitable for trials with patients

Utilization of Organic Synthesis and Spectroscopy in the Investigation of Biological Systems

The investigation of a biological system can be probed in multiple fashions to improve our understanding of how they work. The work presented in this dissertation demonstrates how my work utilizes synthesis and spectroscopy to probe biological systems and gain a deeper understanding of cellular processes. My abilities as both an organic and analytical chemist are displayed throughout the projects that I have worked on throughout my graduate studies. Real advances have been made in the probed biological systems, allowing future researchers to take a more targeted approach from the chemical knowledge presented.Chapter 1 will focus on the degradation of dichloromethane in two members of the Peptococcaceae family. Initial isotope analysis of the degradation of DCM anaerobically suggests that each bacterium have a unique mechanism to convert DCM to non-toxic byproducts. An NMR analysis study using 13C-DCM is applied to both pure culture Dehalobacterium formicoaceticum and the mixed culture consortium RM containing DCM degrader Candidatus Dichloromethanomonas elyunquensis to observe the degradation and subsequently perform a pathway analysis.Chapter 2 is a study on the induction of diabetes that is associated with the prolonged treatment of inflammation with glucocorticoids. These drugs have wide ranging applications due to their global anti-inflammatory properties although they tend to limit the secretion of insulin in β [beta]-cells located in the pancreas. To gain a better understanding of this effect a library of glucocorticoids containing both steroidal and non-steroidal scaffolds were synthesized, characterized, computationally modeled, and tested in-vitro as potential anti-inflammatory drugs with candidates displaying little to no effect on insulin production

Exploring Model-to-Model Transformations for RIA Architectures by means of a Systematic Mapping Study

This study focuses on model-to-model – M2M – transformations, as part of the Model- Driven Development – MDD – approach, for Rich Internet Applications – RIA. The main aim of this study is to identify fields that require further contributions, and/or research opportunities in the previously mentioned context. We applied mapping studies techniques, since these techniques use the same basic methodology as reviews but are more general and aimed at discovering what the research trends are, allowing to identify gaps in the literature. From an initial set of 132 papers, we selected 30 papers first. Then, thanks to experts’ suggestion, we added 3 additional papers. Therefore, we considered 33 research papers. The performed analysis led to various considerations. Among the important ones, we can mention: there are many newly proposed methods, the scarcity of rigorous and formal validation of such methods, the problem of the portability of Platform Independent Models – PIM, and the low number of tools available for MDD.Laboratorio de Investigación y Formación en Informática Avanzad