Application of Liquid Chromatography-Tandem Mass Spectrometry Techniques to the Study of Two Biological Systems

Quorum sensing is a type of bacterial cell-cell communication that uses diffusible signaling molecules to allow the regulation of gene expression based on cell density. The two types of signaling molecules discussed here are autoinducer-2 (AI-2) and a class of signaling molecules of the N-acylhomoserine lactone type (AHL) also known as autoinducer-1 (AI-1). The biosynthesis of both of these molecules has origins in a metabolic pathway. Although undisputed in some cases, the role of AI-2 and AHLs in bacterial systems has come into question. Here, the functionalities of these molecules were investigated by utilizing isotope-labeled versions of both AI-2 and AHLs in conjunction with liquid chromatography tandem mass spectrometry in order to quantify the natural abundance of these molecules in various bacterial cultures. Presented in this thesis are, a synthesis for doubly-deuterated AHLs as well as chromatographic and spectrometric methods for the detection and quantitation of these molecules. Additionally, a series of relevant biological studies which effectively and prolifically utilize these synthetic and analytical techniques are presented here. The selective destruction of beta-cell mass in the Langerhans of the pancreas is known to cause Type 1 diabetes mellitus (T1DM). Currently, many key aspects of this autoimmune disease remain unclear, including the exact mechanism of beta-cell death. In a collaborative project with Dr. J. Jason Collier, we sought to test the hypothesis that different mechanisms of cell death will present discrete phenotypic profiles which can be distinguished by a specific metabolic response in response to the appropriate stimuli. A second project presented in this thesis is the development and implementation of a method to profile the metabolic signatures of two types of pancreatic beta-cell death using tandem mass spectrometry techniques. Using 832/13 rat insulinoma cells, the metabolite pools of cells exposed to either pro-inflammatory cytokines or known apoptosis inducers, such as camptothecin, were analyzed. In this investigation, it was found that this method was effective in defining reproducible metabolic differences in each sample tested. Taken together with complementary methods used in the Collier lab, the results collectively demonstrate that pancreatic beta-cells undergo apoptosis in response to camptothecin, but not pro-inflammatory cytokines

Pancreatic β-Cell Death in Response to Pro-Inflammatory Cytokines Is Distinct from Genuine Apoptosis

A reduction in functional β-cell mass leads to both major forms of diabetes; pro-inflammatory cytokines, such as interleukin-1beta (IL-1β) and gamma-interferon (γ-IFN), activate signaling pathways that direct pancreatic β-cell death and dysfunction. However, the molecular mechanism of β-cell death in this context is not well understood. In this report, we tested the hypothesis that individual cellular death pathways display characteristic phenotypes that allow them to be distinguished by the precise biochemical and metabolic responses that occur during stimulus-specific initiation. Using 832/13 and INS-1E rat insulinoma cells and isolated rat islets, we provide evidence that apoptosis is unlikely to be the primary pathway underlying β-cell death in response to IL-1β+γ-IFN. This conclusion was reached via the experimental results of several different interdisciplinary strategies, which included: 1) tandem mass spectrometry to delineate the metabolic differences between IL-1β+γ-IFN exposure versus apoptotic induction by camptothecin and 2) pharmacological and molecular interference with either NF-κB activity or apoptosome formation. These approaches provided clear distinctions in cell death pathways initiated by pro-inflammatory cytokines and bona fide inducers of apoptosis. Collectively, the results reported herein demonstrate that pancreatic β-cells undergo apoptosis in response to camptothecin or staurosporine, but not pro-inflammatory cytokines. DOI: 10.1371/journal.pone.002248

Pancreatic β-Cell Death in Response to Pro-Inflammatory Cytokines Is Distinct from Genuine Apoptosis

A reduction in functional β-cell mass leads to both major forms of diabetes; pro-inflammatory cytokines, such as interleukin-1beta (IL-1β) and gamma-interferon (γ-IFN), activate signaling pathways that direct pancreatic β-cell death and dysfunction. However, the molecular mechanism of β-cell death in this context is not well understood. In this report, we tested the hypothesis that individual cellular death pathways display characteristic phenotypes that allow them to be distinguished by the precise biochemical and metabolic responses that occur during stimulus-specific initiation. Using 832/13 and INS-1E rat insulinoma cells and isolated rat islets, we provide evidence that apoptosis is unlikely to be the primary pathway underlying β-cell death in response to IL-1β+γ-IFN. This conclusion was reached via the experimental results of several different interdisciplinary strategies, which included: 1) tandem mass spectrometry to delineate the metabolic differences between IL-1β+γ-IFN exposure versus apoptotic induction by camptothecin and 2) pharmacological and molecular interference with either NF-κB activity or apoptosome formation. These approaches provided clear distinctions in cell death pathways initiated by pro-inflammatory cytokines and bona fide inducers of apoptosis. Collectively, the results reported herein demonstrate that pancreatic β-cells undergo apoptosis in response to camptothecin or staurosporine, but not pro-inflammatory cytokines

Prolonged survival in patients with breast cancer and a history of brain metastases: results of a preplanned subgroup analysis from the randomized phase III BEACON trial

Purpose: Conventional chemotherapy has limited activity in patients with breast cancer and brain metastases (BCBM). Etirinotecan pegol (EP), a novel long-acting topoisomerase-1 inhibitor, was designed using advanced polymer technology to preferentially accumulate in tumor tissue including brain metastases, providing sustained cytotoxic SN38 levels. Methods: The phase 3 BEACON trial enrolled 852 women with heavily pretreated locally recurrent or metastatic breast cancer between 2011 and 2013. BEACON compared EP with treatment of physician’s choice (TPC; eribulin, vinorelbine, gemcitabine, nab-paclitaxel, paclitaxel, ixabepilone, or docetaxel) in patients previously treated with anthracycline, taxane, and capecitabine, including those with treated, stable brain metastases. The primary endpoint, overall survival (OS), was assessed in a pre-defined subgroup of BCBM patients; an exploratory post hoc analysis adjusting for the diagnosis-specific graded prognostic assessment (GPA) index was also conducted. Results: In the trial, 67 BCBM patients were randomized (EP, n = 36; TPC, n = 31). Treatment subgroups were balanced for baseline characteristics and GPA indices. EP was associated with a significant reduction in the risk of death (HR 0.51; P < 0.01) versus TPC; median OS was 10.0 and 4.8 months, respectively. Improvement in OS was observed in both poorer and better GPA prognostic groups. Survival rates at 12 months were 44.4% for EP versus 19.4% for TPC. Consistent with the overall BEACON population, fewer patients on EP experienced grade ≥3 toxicity (50 vs. 70%). Conclusions: The significant improvement in survival in BCBM patients provides encouraging data for EP in this difficult-to-treat subgroup of patients. A phase three trial of EP in BCBM patients is underway (ClinicalTrials.gov NCT02915744)

Modeling Runoff and Sediment Yield from a Terraced Watershed Using WEPP

The watershed version of WEPP (Water Erosion Prediction Project) was used to estimate 50-year runoff and sediment yields for a 291 ha watershed in eastern Nebraska that is 90% terraced and which has no historical gage data. The watershed has a complex matrix of elements, including terraced and non-terraced subwatersheds, multiple combinations of soils and land management, a grassed-waterway network, and natural stream channels leading to the outlet. The objectives of this study were to model the study watershed using WEPP and to evaluate model results compared to literature values. WEPP estimated the sediment yield to be 1.9 T/ha/yr, the sediment delivery ratio to be 0.22 and the percent of sediment contribution from the main channel to be 31% of the total sediment yield. These results are consistent with values reported in the literature

Modeling Beaver Dam Effects on Ecohydraulics and Sedimentation in an Agricultural Watershed

Populations of North American beaver (Castor canadensis) have increased in recent decades throughout the agricultural Midwestern U.S., leading to an increase in the frequency of beaver dams in small streams. The impact of beaver dams on channel structure in this region is not known. Our field observations indicate that beaver dams are too dynamic and their affects on channel structure occur over longer time frames than is practical to study with field measurements. Modeling is therefore needed to determine if beaver dams will help stabilize and aggrade incised streams. The objective of this paper is to determine how a channel evolution model (CONCEPTS) might be used to predict the impact of beaver dams on channel structure. The study area, Little Muddy Creek watershed in southeastern Nebraska, is predominantly in agricultural land use. The main reach of the third –order watershed was surveyed for beaver dams from 2003 to 2005. Dam locations were mapped, integrity of dam structure was noted, and water surface elevations were measured. Failure of dam structure was documented following runoff-producing storms. While some dams were repaired within weeks, others were abandoned and left to degrade, causing a significant and transient change in the water surface profile of the stream. Field observations showed that the spatial arrangement and hydraulic condition of beaver dams were temporally dynamic in both short and long term scales. Laboratory tests were conducted to determine if beaver dams could be modeled as broad-crested weirs. Discharge-rating curves were developed for a simulated beaver dam and a conventional weir. The roughness caused by the sticks on the surface of the dam significantly altered the stage-discharge relationship, but did not violate the broad-crested weir model. Accounting for the temporal dynamics of spatial and hydraulic characteristics of beaver dams represents the greatest challenge to modeling the impact of beaver dams on stream channel morphology. TheCONCEPTS model, however, enables manually inserting the appropriate temporal patterns of structural change into model simulations and, thus, allows prediction of dynamic, long-term effects

FLOW PATHWAYS AND SEDIMENT TRAPPING IN A FIELD-SCALE VEGETATIVE FILTER

Vegetative filters (VF) are a best management practice installed in many areas to control sediment movement to water bodies. It is commonly assumed that runoff proceeds perpendicularly across a VF as sheet flow. However, there is little research information on natural pathways of water movement and performance of field-scale VF. The objectives of this study were: (1) to quantify the performance of a VF where the flow path is not controlled by artificial borders and flow path lengths are field-scale, and (2) to develop methods to detect and quantify overland flow convergence and divergence in a VF. Our hypothesis is that flow converges and diverges in field-scale VF and that flow pathways that define flow convergence and divergence areas can be predicted using high-resolution topography (i.e., maps). Overland flow and sediment mass flow were monitored in two 13 × 15 m subareas of a 13 × 225 m grass buffer located in Polk County in east-central Nebraska. Monitoring included a high-resolution survey to 3 cm resolution, dye tracer studies to identify flow pathways, and measurement of maximum flow depths at 51 points in each subarea. Despite relatively planar topography (a result of grading for surface irrigation), there were converging and diverging areas of overland flow in the buffer subareas. Convergence ratios ranged from −1.55 to 0.34. Predicted flow pathways using the high-resolution topography (i.e., map) closely followed actual flow paths. Overland flow was not uniformly distributed, and flow depths were not uniform across the subareas. Despite converging and diverging flow, the field-scale VF trapped approximately 80% of the incoming sediment