State of Nev. Local Gov’t Emp. Mgmt. Bd., v. Educ. Support Emp. Ass’n, 134 Nev. Adv. Op. 86 (Nov. 8, 2018)

The Court determined that the plain language application of NRS § 288.160 and Nevada Administrative Code (NAC) 288.110 states that the vote-counting standard is to be determined by the majority of members of the bargaining unit and not by a majority of the votes cast

Chlorhexidine Substantivity on Salivary Flora and Plaque-Like Biofilm: An In Situ Model

This work was supported by project FIS PI11/01383 from Carlos III Institute of Health (Ministry of Economy and Competitiveness, Madrid, Spain

Antiplaque Effect of Essential Oils and 0.2% Chlorhexidine on an In Situ Model of Oral Biofilm Growth: A Randomised Clinical Trial

This work was supported by project PI11/ 01383 from Carlos III Institute of Health (General Division of Evaluation and Research Promotion, Madrid, Spain), which is integrated in National Plan of Research, Development and Innovation (PN I+D+I 2008-2011). This project was cofinanced by European Regional Development Fund (ERDF 2007- 2013

Beyond structural models for the mode of action:How natural antimicrobial peptides affect lipid transport

Hypothesis: Most textbook models for antimicrobial peptides (AMP) mode of action are focused on structural effects and pore formation in lipid membranes, while these deformations have been shown to require high concentrations of peptide bound to the membrane. Even insertion of low amounts of peptides in the membrane is hypothesized to affect the transmembrane transport of lipids, which may play a key role in the peptide effect on membranes. Experiments: Here we combine state-of-the-art small angle X-ray/neutron scattering (SAXS/SANS) techniques to systematically study the effect of a broad selection of natural AMPs on lipid membranes. Our approach enables us to relate the structural interactions, effects on lipid exchange processes, and thermodynamic parameters, directly in the same model system. Findings: The studied peptides, indolicidin, aurein 1.2, magainin II, cecropin A and LL-37 all cause a general acceleration of essential lipid transport processes, without necessarily altering the overall structure of the lipid membranes or creating organized pore-like structures. We observe rapid scrambling of the lipid composition associated with enhanced lipid transport which may trigger lethal signaling processes and enhance ion transport. The reported membrane effects provide a plausible canonical mechanism of AMP-membrane interaction and can reconcile many of the previously observed effects of AMPs on bacterial membranes

Oral Biofilm Architecture on Natural Teeth

Periodontitis and caries are infectious diseases of the oral cavity in which oral biofilms play a causative role. Moreover, oral biofilms are widely studied as model systems for bacterial adhesion, biofilm development, and biofilm resistance to antibiotics, due to their widespread presence and accessibility. Despite descriptions of initial plaque formation on the tooth surface, studies on mature plaque and plaque structure below the gum are limited to landmark studies from the 1970s, without appreciating the breadth of microbial diversity in the plaque. We used fluorescent in situ hybridization to localize in vivo the most abundant species from different phyla and species associated with periodontitis on seven embedded teeth obtained from four different subjects. The data showed convincingly the dominance of Actinomyces sp., Tannerella forsythia, Fusobacterium nucleatum, Spirochaetes, and Synergistetes in subgingival plaque. The latter proved to be new with a possibly important role in host-pathogen interaction due to its localization in close proximity to immune cells. The present study identified for the first time in vivo that Lactobacillus sp. are the central cells of bacterial aggregates in subgingival plaque, and that Streptococcus sp. and the yeast Candida albicans form corncob structures in supragingival plaque. Finally, periodontal pathogens colonize already formed biofilms and form microcolonies therein. These in vivo observations on oral biofilms provide a clear vision on biofilm architecture and the spatial distribution of predominant species

Studies on the evolution of the adaptive immune system

T Lymphozyten sind der zentrale Zelltyp des zellulären Arms des adaptiven Immunsystems der Gnathostomata (Kiefermäuler) und erkennen ihr Antigen in Form kurzer Oligopeptide, die von antigenpräsentierenden Zellen an Haupthistokompatibilitäts-Komplex (MHC)-Moleküle gebunden präsentiert werden. T Lymphozyten erzeugen im Laufe ihrer Entwicklung aus T-Zell-Vorläufern unikale Antigenrezeptor-Gene durch Rekombination verschiedener Gensegmente, weshalb sie einen T Zell Rezeptor (TCR) zufälliger Spezifität tragen. Dies macht die Kopplung eines Selektionsprozesses an die T-Zell-Entwicklung notwendig. In Organismen mit adaptivem Immunsystem übernimmt der Thymus diese Funktion, ein spezialisiertes Organ, in dem das T-Zell-Entwicklungsprogramm räumlich an die Qualitätskontrolle der entstehenden T Lymphozyten gekoppelt ist. Im Rahmen dieser Arbeit wurden drei Projekte bearbeitet, die sich mit der Evolution dieses Qualitätskontrollsystems beschäftigen: Der Transkriptionsfaktor Foxn1 (Forkhead box N1) ist essentiell für die Ontogenese des Thymus, in Abwesenheit von Foxn1 bricht die Thymusentwicklung ab, da das entstehende Thymusepithel keine T-Zell-Vorläufer attrahieren kann. Auch in der Phylogenese des Thymus scheint Foxn1 eine wichtige Rolle zu spielen, da das Auftreten von Foxn1 und des Thymus im Laufe der Evolution der Chordaten zusammenfallen. Im ersten Teil dieser Arbeit wurde die evolutionäre Geschichte von Foxn1 untersucht. Durch Vergleich der Aminosäuresequenz, der Syntenie und der Intronkonfiguration Foxn1-artiger Gene verschiedener Organismen konnte gezeigt werden, dass Foxn1 sich nach einer Genduplikation aus einem Foxn4-Paralog entwickelt hat. Zudem wurde die Expression verschiedener Foxn1-artiger Gene und eines von Foxn4 regulierten Gens im Neunauge, im Hai und im Huhn untersucht. Mit den Ergebnissen dieser und anderer parallel durchgeführter Untersuchungen konnte ein Modell entwickelt werden, nach dem die Transkriptionskontrolle über das Chemokin-Gen CCL25 die entscheidende Eigenschaft ist, die es Foxn1 ermöglicht, die Thymusentwicklung zu steuern. Im zweiten Teil sollte Foxn1 unter der Kontrolle des Glial cells missing homolog 2 (Gcm2)-Promotors in der Nebenschilddrüsen-Organanlage exprimiert werden, um Erkenntnisse über die Rolle von Foxn1 bei der Organogenese des Thymus und der Embryonalentwicklung der dritten Schlundtasche zu erlangen. Hierfür wurden durch homologe Rekombination in ES-Zellen und anschließende Blastozysten-Injektion zwei Gcm2::Foxn1 Knock-in Mauslinien erzeugt. Bis zur Fertigstellung dieser Arbeit konnte bei diesen Tieren jedoch kein Foxn1-Transkript in den Zellen der Nebenschilddrüse nachgewiesen werden. Der dritte Teil dieser Arbeit befasst sich mit der Evolution der MHC-Peptidpräsentation. Da diese die Grundlage für die Antigenerkennung durch T Lymphozyten und somit für die Qualitätskontrolle der TCRs darstellt, liegt es nahe zu postulieren, dass bereits vor der Entstehung der T Zellen und des adaptiven Immunsystems der Gnathostomata ein biologischer Mechanismus existiert haben muss, der für die Antigenpräsentation adaptiert werden konnte. MHC-Peptide zeigen nicht nur den Infektionsstatus von Zellen an, sondern enthalten in den charakteristischen Ankeraminosäuren auch Information über den Genotyp des Organismus, der sie erzeugt hat. Daher wurde die Hypothese aufgestellt, dass der Vorläufer des MHC-Peptidpräsentationssystems eine Rolle bei der Wahl des Fortpflanzungspartners gespielt hat. Voraussetzung hierfür ist allerdings die Existenz eines Mechanismus, welcher es ermöglicht, die in den MHC-Peptiden enthaltene Genotyp-Information auszulesen. Die im basalen Teil des sensorischen Epithels des Vomeronasalorgans der Maus gelegenen Neuronen (VSNs) erkennen spezifisch MHC-Peptide, was einen solchen Mechanismus darstellt, und somit die oben formulierte Hypothese stützt. Um zu zeigen, dass die G-Protein gekoppelten Rezeptoren des V2R-Typs, welche von diesen VSNs exprimiert werden, die spezifische Erkennung von MHC-Peptiden vermitteln, wurden die extrazellulären Domänen ausgewählter V2Rs in einem zellfreien Expressionssystem erzeugt, aufgereinigt und auf MHC-Peptidbindung untersucht. Mit der verwendeten Methode konnte jedoch keine spezifische Erkennung von MHC-Peptiden nachgewiesen werden.T lymphocytes play a central role in cell-mediated immune responses of the adaptive immune system in jawed vertebrates. They recognize their antigen as short oligopeptides that are bound to Major Histocompatibility (MHC)-molecules on antigen-presenting cells (APCs). During their development from bone-marrow derived progenitors, each T lymphocyte generates unique antigen-receptor genes by joining pre-existing gene segments in a process called V(D)J recombination, resulting in a T cell receptor (TCR) of random specificity. Consequently, developing T cells need to be screened and selected for functionality and self-tolerance of their TCR. In jawed vertebrates, a specialized organ has evolved, that spatially couples this quality control process to the T lymphocyte development program - the thymus. This PhD thesis integrates the results from three projects revolving around the evolution of this quality control system: The forkhead box transcription factor Foxn1 is crucial for the ontogeny of the thymus: In its absence, thymus development is blocked, as the developing thymic epithelium fails to attract lymphocyte progenitors. Foxn1 seems to play an important role in the phylogeny of the thymus as well, since the emergence of Foxn1 and the thymus coincide during the course of chordate evolution. The first topic addressed in this thesis is the evolutionary history of Foxn1. By comparing protein sequences, synteny relationships and intron configurations of Foxn1-like genes (Foxn1, Foxn4 and the novel Foxn4-like gene) from a number of species, it could be shown that Foxn1 evolved from a paralog of the closely related Foxn4 gene after a gene duplication event in the common ancestor of the vertebrates. In addition, the expression patterns of several Foxn1-like genes and of a Foxn4-target gene were examined in lamprey, catshark and chicken. The results of this study and of experiments that were conducted in parallel led to the development of a model of thymus phylogeny which states that the key feature of Foxn1 that allows it to drive thymus development is its transcriptional control over the chemokine gene CCL25. The goal of the second part of this thesis was to express Foxn1 in the parathyroid anlage under the control of the Glial cells missing homolog 2 (Gcm2)-promoter, in order to gain insights into the role of Foxn1 in thymus organogenesis and the embryonic development of the third pharyngeal pouch. To this end, two Gcm2::Foxn1 Knock-in mouse strains were generated via homologous recombination in mouse ES cells. However, no animals with Foxn1-transcripts in parathyroid cells could be identified by the time this thesis was completed. The final part of this thesis addresses the evolution of MHC-peptide presentation. Given its crucial function in antigen recognition by and therefore quality control of T cells, it can be postulated that a biological mechanism that could be adapted for antigen presentation must have existed before the emergence of the adaptive immune system of the jawed vertebrates. Besides indicating the health status of the cell presenting them, MHC peptides - in the form of their anchor residues - contain information about the genotype of the individual in which they were created. Thus it was hypothesized that the ancestor of the MHC-peptide presentation system had a function in mate choice, which necessitates the existence of a mechanism that allows the read-out of the genotype information contained in MHC peptides. In mice, the vomeronasal sensory neurons (VSNs) in the basal part of the Vomeronasal Organ’s sensory epithelium specifically detect MHC peptides by their anchor residues, which constitutes such a mechanism, supporting above-mentioned hypothesis. In order to show that the G protein coupled receptors of the V2R type that are expressed by these VSNs mediate the specific recognition of MHC peptides, the extracellular domains of selected V2Rs were produced in a cell-free expression system, purified and tested for MHC-peptide binding. The experimental approach used here did not lead to the detection of anchor residue-specific binding of MHC peptides however