The Discovery, Characterization, and Engineering of Autolytic Enzymes as Antimicrobials

In an age of rising levels of antibiotic resistance, we are quickly running out of tools to address some of the most virulent and widespread infectious bacteria. One group of enzymes that show significant potential for use as next-generation antibiotics are antimicrobial peptidoglycan hydrolases, often referred to as lysins. These enzymes are responsible for the breakdown of peptidoglycan within the bacterial cell wall, and, when exogenously applied, can result in dramatic destabilization of the cell wall leading to rapid lysis of the bacteria. While phage endolysins have been widely investigated as potential antimicrobials, the therapeutic potential of endogenous autolysins from pathogenic bacteria is relatively unexplored. This thesis focuses on three projects, the first two are aimed at the identification, characterization, and engineering of genomically derived autolysins with antimicrobial activity against Clostridioides difficile and Staphylococcus aureus, two pathogens with concerning antibiotic resistance, while the final project pushes the development of techniques to address a common issue for bacterial lysins, immunogenicity in the form of antidrug antibodies. The first project details the bioinformatics-driven discovery of six new anti-C. difficile lysins belonging to the amidase-3 family of enzymes, and we describe experimental comparison of these new variants against the leading candidates from the literature. Our quantitative analyses include metrics for expression level, inherent antibacterial activity, breadth of strain selectivity, killing of germinating spores, and structural and functional measures of thermal stability. In the second project we combine a bioinformatics pipeline to identify putative Staphylococcus aureus autolysin domains, with a combinatorial approach to generate a library of chimeric autolysin which were subsequently screened in a high-throughput manner resulting in the discovery and characterization of the novel antimicrobial chimeric autolysin Ssa2-301. In the third project we focus on utilizing a technique to eliminate B cell epitopes from the surface of lytic enzymes using the previously characterized bacteriocin lysostaphin as a proof of principle. These projects illustrate the value of expanding efforts in the exploration of autolysins as potential antimicrobials, and they illustrate one example of the often-necessary engineering to improve their translational potential

Geographic Opportunity and Neomalthusian Willingness: Boundaries, Shared Rivers, and Conflict

International conflict has been analyzed extensively through the framework of opportunity and willingness. Opportunity has mainly been operationalized as physical proximity. Willingness has been measured in a number of ways, and remains a somewhat more elusive concept. Several scholars have called for boundary length to represent opportunity. Heeding such calls, Harvey Starr has used GIS methods to generate boundary length for 1993 and has found it to be associated with increased propensity to conflict. A number of his measures of willingness were not. Using a new and much more extensive dataset on boundary length for the entire Correlates of War period, this article finds very different results. We study the relationship with shared rivers and water scarcity as measures of neomalthusian factors in willingness over a 110-year period. The results indicate that the neomalthusian factors are significant although not dramatic in their effects. Boundary length, while associated with conflict in a bivariate analysis, fades into insignificance when the neomalthusian willingness measures are introduced

Geographic Opportunity and Neomalthusian Willingness: Boundaries, Shared Rivers, and Conflict

International conflict has been analyzed extensively through the framework of opportunity and willingness. Opportunity has mainly been operationalized as physical proximity. Willingness has been measured in a number of ways, and remains a somewhat more elusive concept. Several scholars have called for boundary length to represent opportunity. Heeding such calls, Harvey Starr has used GIS methods to generate boundary length for 1993 and has found it to be associated with increased propensity to conflict. A number of his measures of willingness were not. Using a new and much more extensive dataset on boundary length for the entire Correlates of War period, this article finds very different results. We study the relationship with shared rivers and water scarcity as measures of neomalthusian factors in willingness over a 110-year period. The results indicate that the neomalthusian factors are significant although not dramatic in their effects. Boundary length, while associated with conflict in a bivariate analysis, fades into insignificance when the neomalthusian willingness measures are introduced

Electrostatic-Mediated Affinity Tuning of Lysostaphin Accelerates Bacterial Lysis Kinetics and Enhances <i>In Vivo</i> Efficacy

Drug-resistant bacterial pathogens are a serious threat to global health, and antibacterial lysins are at the forefront of innovative treatments for these life-threatening infections. While lysins’ general mechanism of action is well understood, the design principles that might enable engineering of performance-enhanced variants are still being formulated.</jats:p

Separação e quantificação de riboflavina em leite desnatado UAT por UHPLC-FL

Esse estudo visou estabelecer as condições de separação e quantificação da riboflavina em amostras de leite ultra alta temperatura (UAT) desnatado. As análises foram realizadas por meio de extração ácida seguido de separação e quantificação por cromatografia líquida de ultra alta eficiência acoplado de detector de fluorescência (UHPLC-FL). Foram ainda testados a linearidade e a seletividade do método através da construção das curvas de calibração e de efeito de matriz, respectivamente. A riboflavina foi detectada em tempo de retenção (tr) de 6,35 minutos e ambas as curvas apresentaram coeficientes de determinação (r2) acima de 0,99, além de o método se apresentar seletivo, por isso o mesmo foi considerado eficaz para a determinação de riboflavina em amostras de leite UAT desnatado.</jats:p