New Techniques for the Qualitative and Quantitative Measurement of Naturally-Ocurring Gonadotropin-Releasing Hormone Analogues by Mass Spectrometry

GnRH peptides have been discovered in a wide variety of vertebrate and invertebrate organisms, and work is ongoing to characterize additional unique isoforms. This dissertation describes the investigation of reversed-phase chromatographic and mass spectrometric behavior of GnRH peptides, the development and application of an LC-MS/MS method for qualitative identification of GnRH peptides, and the comprehensive validation of an LC-MS/MS method for simultaneous, quantitative measurement of hydroxyproline9GnRH (Hyp9GnRH) and mammalian GnRH (mGnRH) in rat brain tissues. Chromatographic and mass spectrometric behavior of GnRH isoforms was characterized for six GnRH model peptides. Using reversed-phase high performance liquid chromatography (HPLC), nearly complete separation of the model GnRH peptides was achieved. Evaluation of electrospray source conditions indicated that certain parameters can be adjusted to affect the abundance of selected charge states and improve response. Using the conditions found to be optimal for GnRH peptides in general, a method was developed to facilitate characterization of novel GnRH isoforms or confirm the identity of known isoforms. Fragmentation patterns for six model GnRH isoforms were examined to determine what portion of the primary sequence could be elucidated by de novo sequencing, and a simple solid phase extraction protocol was developed to isolate the model GnRH compounds from tissue samples. Application of the method to rat brain samples resulted in successful isolation and structural confirmation of hydroxyproline9GnRH and mammalian GnRH. A quantitative method for the determination of concentrations of hydroxyproline9GnRH and mammalian GnRH in rat brain tissue was developed and rigorously validated. Guinea pig brains were found to be a suitable substitute matrix for rat brains, and accuracy and precision were determined after four validation runs. Stability of both peptides in samples over long-term storage and under experimental conditions were evaluated, and the LC-MS/MS method was compared to an enzyme-linked immunoassay. Thirty-one brains from Sprague-Dawley rats were analyzed using the LC-MS/MS procedure and compared to published results for Hyp9GnRH and mGnRH

Fecal Transplantation does not Transfer Either Susceptibility or Resistance to Food Borne Listeriosis in C57BL/6 and BALB/c/By Mice

The composition of the intestinal microbiota has wide reaching effects on the health of an individual, including the development of protective innate immune responses. In this report, a fecal transplantation approach was used to determine whether resistance to food borne listeriosis was dependent on the murine gut microbiota. Transplantation of BALB/c/By feces did not increase the susceptibility of C57BL/6 mice to Listeria monocytogenes infection. Likewise, transplantation of C57BL/6 fecal matter did not enhance the resistance of BALB/c/By mice. Thus, intestinal microbiota composition is not a key factor that confers either susceptibility or resistance to food borne listeriosis in mice

Oral Transmission of Listeria Monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection

Composition and Methods for Treating \u3cem\u3eYersinia Pestis\u3c/em\u3e Infection

Compositions and methods for treating a Yersinia pestis (Y. pestis) infection are provided. Compositions and methods of for inducing an immune response in a subject are provided. Composition can include a YadC polypeptide

Calibration Systems Final Report

The Calibration Systems project at Pacific Northwest National Laboratory (PNNL) is aimed towards developing and demonstrating compact Quantum Cascade (QC) laser-based calibration systems for infrared imaging systems. These on-board systems will improve the calibration technology for passive sensors, which enable stand-off detection for the proliferation or use of weapons of mass destruction, by replacing on-board blackbodies with QC laser-based systems. This alternative technology can minimize the impact on instrument size and weight while improving the quality of instruments for a variety of missions. The potential of replacing flight blackbodies is made feasible by the high output, stability, and repeatability of the QC laser spectral radiance

Broadly Tunable External Cavity Quantum Cascade Laser Development

FY07 final report for Laser Development task of ITAS project

Intracellular \u3cem\u3eListeria monocytogenes\u3c/em\u3e Comprises a Minimal but Vital Fraction of the Intestinal Burden Following Foodborne Infection

Listeria monocytogenes is a highly adaptive bacterium that replicates as a free-living saprophyte in the environment as well as a facultative intracellular pathogen that causes invasive foodborne infections. The intracellular life cycle of L. monocytogenes is considered to be its primary virulence determinant during mammalian infection; however, the proportion of L. monocytogenes that is intracellular in vivo has not been studied extensively. In this report, we demonstrate that the majority of wild-type (strain EGDe) and mouse-adapted (InlAm-expressing) L. monocytogenes recovered from the mesenteric lymph nodes (MLN) was extracellular within the first few days after foodborne infection. In addition, significantly lower burdens of L. monocytogenes were recovered from the colon, spleen, and liver of gentamicin-treated mice than of control mice. This led us to investigate whether intracellular replication of L. monocytogenes was essential during the intestinal phase of infection. We found that lipoate protein ligase-deficient L. monocytogenes (ΔlplA1) mutants, which display impaired intracellular growth, were able to colonize the colon but did not persist efficiently and had a significant defect in spreading to the MLN, spleen, and liver. Together, these data indicate that the majority of the L. monocytogenes burden in the gastrointestinal tract is extracellular, but the small proportion of intracellular L. monocytogenes is essential for dissemination to the MLN and systemic organs

InlA promotes dissemination of Listeria monocytogenes to the mesenteric lymph nodes during food borne infection of mice

Intestinal Listeria monocytogenes infection is not efficient in mice and this has been attributed to a low affinity interaction between the bacterial surface protein InlA and E-cadherin on murine intestinal epithelial cells. Previous studies using either transgenic mice expressing human E-cadherin or mouse-adapted L. monocytogenes expressing a modified InlA protein (InlA(m)) with high affinity for murine E-cadherin showed increased efficiency of intragastric infection. However, the large inocula used in these studies disseminated to the spleen and liver rapidly, resulting in a lethal systemic infection that made it difficult to define the natural course of intestinal infection. We describe here a novel mouse model of oral listeriosis that closely mimics all phases of human disease: (1) ingestion of contaminated food, (2) a distinct period of time during which L. monocytogenes colonize only the intestines, (3) varying degrees of systemic spread in susceptible vs. resistant mice, and (4) late stage spread to the brain. Using this natural feeding model, we showed that the type of food, the time of day when feeding occurred, and mouse gender each affected susceptibility to L. monocytogenes infection. Co-infection studies using L. monocytogenes strains that expressed either a high affinity ligand for E-cadherin (InlA(m)), a low affinity ligand (wild type InlA from Lm EGDe), or no InlA (ΔinlA) showed that InlA was not required to establish intestinal infection in mice. However, expression of InlA(m) significantly increased bacterial persistence in the underlying lamina propria and greatly enhanced dissemination to the mesenteric lymph nodes. Thus, these studies revealed a previously uncharacterized role for InlA in facilitating systemic spread via the lymphatic system after invasion of the gut mucosa

Latinos in Sampson County, North Carolina : an action-oriented community diagnosis

The following document is a detailed report of an Action Oriented Community Diagnosis (AOCD) conducted by a team of six students from the University of North Carolina Chapel Hill, School of Public Health, Department of Health Behavior and Health Educations in collaboration with the Sampson County Health Department. The AOCD was conducted in order to understand the cultural, social, economic, and health experiences of Latinos living in or accessing services in Sampson County. Throughout the AOCD process, the AOCD student team and community participants worked together to identify the strengths and challenges of Latinos living or accessing services in Sampson County and to create action steps addressing several of the identified challenges. Between September 2007 and April 2008, with the help of community liaisons from the Sampson County Health Department, the student team made an effort to learn about the community. By reviewing secondary sources of information such as newspapers, and websites, conducting in-depth interviews with 16 service providers and nine community members; and facilitating five focus groups with community members, the team members gained a broad perspective on issues important to the Latino community in Sampson County. The team organized a data coding system to identify recurring themes related to the Latino community living in or accessing services in Sampson County. On April 19, 2008, the analyzed data were presented to the community at a forum held at the Sampson Community College, with the aim of bridging different viewpoints, creating a dialogue among county residents and service providers, and developing actions steps to address the identified challenges. The specific challenges included transportation, language and communication, awareness of services, leadership, housing, and recreation. The following are action steps that resulted from the group discussion at the forum: Raise awareness of transportation services that currently exist in Sampson County through Spanish language newspapers and radio. Get more people involved in community events. Organize ongoing community meetings to share information between service providers and community members. Following the forum, the student team compiled this report that presents recommendations for the community based on results from the forum and its experiences in Sampson County. The principal final recommendations include: The team recommends that service providers and community members work closely together to develop materials and to organize events that are culturally appropriate and will encourage more Latinos to participate in community discussions. The team also recommends that advertisements for activities, events or services clearly state whether translation services are provided or bilingual staff will be present and what, if any, documentation is required. Further, it is recommended that these advertisements are distributed or announced in a manner that is likely to reach the Latino population, (e.g., door-to-door flier circulation, Spanish language radio, Spanish language newspapers, etc.) The team strongly recommends that service providers work in collaboration with community members in any community organizing effort in order to best serve the community‘s needs. Following the action steps identified, the team recommends that ongoing community meetings are held in an effort to bring together service providers and community members to share information. The student team hopes that this document and its contents will serve as a resource for continuing a community dialogue initiated at the community forum. Ultimately, the team hopes that the entire AOCD process and final report will lead to improvements not only for Latinos, but also for all residents of Sampson County.Master of Public Healt

Cyclic di-GMP-dependent Signaling Pathways in the Pathogenic Firmicute Listeria Monocytogenes

We characterized key components and major targets of the c-di-GMP signaling pathways in the foodborne pathogen Listeria monocytogenes, identified a new c-di-GMP-inducible exopolysaccharide responsible for motility inhibition, cell aggregation, and enhanced tolerance to disinfectants and desiccation, and provided first insights into the role of c-di-GMP signaling in listerial virulence. Genome-wide genetic and biochemical analyses of c-di-GMP signaling pathways revealed that L. monocytogenes has three GGDEF domain proteins, DgcA (Lmo1911), DgcB (Lmo1912) and DgcC (Lmo2174), that possess diguanylate cyclase activity, and three EAL domain proteins, PdeB (Lmo0131), PdeC (Lmo1914) and PdeD (Lmo0111), that possess c-di-GMP phosphodiesterase activity. Deletion of all phosphodiesterase genes (ΔpdeB/C/D) or expression of a heterologous diguanylate cyclase stimulated production of a previously unknown exopolysaccharide. The synthesis of this exopolysaccharide was attributed to the pssA-E (lmo0527-0531) gene cluster. The last gene of the cluster encodes the fourth listerial GGDEF domain protein, PssE, that functions as an I-site c-di-GMP receptor essential for exopolysaccharide synthesis. The c-di-GMP-inducible exopolysaccharide causes cell aggregation in minimal medium and impairs bacterial migration in semi-solid agar, however, it does not promote biofilm formation on abiotic surfaces. The exopolysaccharide also greatly enhances bacterial tolerance to commonly used disinfectants as well as desiccation, which may contribute to survival of L. monocytogenes on contaminated food products and in food-processing facilities. The exopolysaccharide and another, as yet unknown c-di-GMP-dependent target, drastically decrease listerial invasiveness in enterocytes in vitro, and lower pathogen load in the liver and gallbladder of mice infected via an oral route, which suggests that elevated c-di-GMP levels play an overall negative role in listerial virulence