Expression and proteolytic cleavage of Lav, a putative virulence autotransporter of nontypeable Haemophilus influenzae [abstract]

Abstract only availableHaemophilus influenzae, a gram negative coccobacillus, is part of the normal flora of the human upper respiratory tract. Unencapsulated (nontypeable) strains of H. influenzae (NTHi) cause pneumonia, sinusitis, and otitis media, and complicate chronic lung disease. Strain R2866 is unusually invasive and was isolated from a child with meningitis. R2866 and many other NTHi have a gene termed lav, whose encoded protein belongs to the family of virulence-associated autotransporters. A nearly identical gene is found in the meningococcus, which obtained it by horizontal transmission from H. influenzae. Lav is a phase-variable outer membrane protein, with the ON or OFF phase controlled by the number of tetranucleotide repeats downstream of the initiating codon. We are investigating the function and localization of Lav. Most H. influenzae autotransporters are adhesins. Lav is not a primary adhesin, but preliminary experiments suggest it may improve adherence to human lung tissue cells. Autotransporters consist of three primary domains, an N-terminal signal peptide, a C-terminal beta-barrel domain that forms a pore in the outer membrane, and a middle "passenger" domain that is the effector part of the protein and is exported through the pore. In most autotransporters, the "passenger" domain is proteolytically cleaved from the beta-domain, usually remaining bound at the cell surface. We are testing to see whether Lav is similarly cleaved, by comparing whole cell extracts, outer membrane fractions, and culture supernatants. Cell fractions are Western-blotted and probed with antibodies specific for the passenger domain or the C-terminus. Strains include R2866 with Lav in the ON and OFF phase, a phase-locked ON derivative of R2866, a null Lav mutant, and E coli and H. influenzae Rd engineered to express Lav. A 20-kDa fragment with the passenger N-terminal epitope was found in culture supernatants of strains expressing Lav but not in those of control non-expressing strains

Autoaggregation and adhesion by nontypeable Haemophilus influenzae strains with mutations in the autotransporter Lav

Abstract only availableNontypeable Haemophilus influenzae (NTHi) is a nonencapsulated gram-negative coccobacillus that colonizes the upper respiratory tract of most healthy adults. Pathogenic NTHi cause otitis media, sinusitis, bronchitis, pneumonia, and complicate COPD and cystic fibrosis. Many NTHi from clinical isolates (but not commensal NTHi) encode Lav, homologous to bacterial autotransporter proteins. Lav is a phase-variable outer membrane protein whose expression is ON or OFF depending on the number of GCAA repeats following the initiating ATG codon. Preliminary experiments suggested that Lav improves adherence to lung tissue culture cells, but is not a primary adhesin. As some adhesins affect bacterial autoaggregation, a component of biofilm formation, we compared mutants with a knockout mutation in Lav to wild-type and phase-locked ON strains and found that null mutants caused unusually rapid autoaggregation of the null mutant relative to strains expressing Lav. However, the rapidly aggregating null mutant was seen to form filaments, leading us to suspect that its construction affected expression of neighboring genes (e.g., tmk) essential for DNA synthesis. Starting with the phase-locked ON construct, I constructed new mutants deleted for Lav that were fully isogenic with positive control strains. We will repeat autoaggregation and cell adherence assays using these more appropriate knockout mutants.NSF-REU Program in Biological Sciences & Biochemistr

Freiheit als Kritik. Zur Debatte um Freiheit bei Foucault

In der Debatte um Freiheit bei Foucault wird das „Freiheitsproblem“ verhandelt: Wie können Freiheit und Widerstand innerhalb von Foucaults Theorie der Macht und Subjektivierung konzipiert werden? Der Aufsatz leistet eine Rekonstruktion und interne Kritik der besten Interpretationsstrategien von Foucaults Werk, die die Lösung dieses Problems zum Ziel haben, und entwickelt dabei eine neue These: Freiheit als Fähigkeit zur reflexiven Kritik der eigenen Subjektivierung ist abhängig von freiheitlicher Subjektivierung durch politische Institutionen. Die Interpretationsstrategien werden systematisch unterschieden und anhand der Arbeiten exemplarischer Vertreter_innen diskutiert: 1. Foucault ist kohärent, 2. Foucault korrigiert sich, 3. Foucault kritisiert kohärent, 4. Foucault ist nicht genug. Gegen Lemke wird argumentiert, dass Freiheit nicht durch eine anarchistische, sondern nur durch eine institutionalistische Foucault-Lesart bestimmt werden kann. Mit Saar wird gezeigt, dass das Ziel einer eindeutigen Definition von Freiheit im Gegensatz zur Methode der genealogischen Kritik steht und nur im Rahmen der normativen politischen Theorie verfolgt werden kann. In Auseinandersetzung mit Allen wird argumentiert, dass Freiheit nicht in linken Bewegungen verortet werden kann, sondern nur in demokratischen Institutionen

Banned Books Week: Big Ideas and Freedom of Information

Banned Books Week: Big Ideas and Freedom of Information Presented by Members of Sigma Tau Delta English Honor Society Taylor Vredenburg, Isaiah Russell, Harrison Jenkins, and CJ Gibson October 4, 202

The Quick-Freezing of Single Intact Skeletal Muscle Fibers at Known Time Intervals Following Electrical Stimulation

Single intact frog skeletal muscle fibers quick-frozen after known time intervals following electrical stimulation are examined electron microscopically in thin sections, after freeze-substitution, in freeze-fracture/etch preparations, and in cryosections prepared for x-ray microprobe analysis. Techniques are described to perform these operations on a single fiber. Notable morphological differences between conventionally fixed and cryopreserved muscle fibers, and between fibers quick- frozen at different post-stimulation intervals are demonstrated

A computational framework for generating patient-specific vascular models and assessing uncertainty from medical images

Patient-specific computational modeling is a popular, non-invasive method to answer medical questions. Medical images are used to extract geometric domains necessary to create these models, providing a predictive tool for clinicians. However, in vivo imaging is subject to uncertainty, impacting vessel dimensions essential to the mathematical modeling process. While there are numerous programs available to provide information about vessel length, radii, and position, there is currently no exact way to determine and calibrate these features. This raises the question, if we are building patient-specific models based on uncertain measurements, how accurate are the geometries we extract and how can we best represent a patient's vasculature? In this study, we develop a novel framework to determine vessel dimensions using change points. We explore the impact of uncertainty in the network extraction process on hemodynamics by varying vessel dimensions and segmenting the same images multiple times. Our analyses reveal that image segmentation, network size, and minor changes in radius and length have significant impacts on pressure and flow dynamics in rapidly branching structures and tapering vessels. Accordingly, we conclude that it is critical to understand how uncertainty in network geometry propagates to fluid dynamics, especially in clinical applications.Comment: 21 pages, 9 figure

In search of the authentic nation: landscape and national identity in Canada and Switzerland

While the study of nationalism and national identity has flourished in the last decade, little attention has been devoted to the conditions under which natural environments acquire significance in definitions of nationhood. This article examines the identity-forming role of landscape depictions in two polyethnic nation-states: Canada and Switzerland. Two types of geographical national identity are identified. The first – what we call the ‘nationalisation of nature’– portrays zarticular landscapes as expressions of national authenticity. The second pattern – what we refer to as the ‘naturalisation of the nation’– rests upon a notion of geographical determinism that depicts specific landscapes as forces capable of determining national identity. The authors offer two reasons why the second pattern came to prevail in the cases under consideration: (1) the affinity between wild landscape and the Romantic ideal of pure, rugged nature, and (2) a divergence between the nationalist ideal of ethnic homogeneity and the polyethnic composition of the two societies under consideration