The Burkholderia cenocepacia Type VI Secretion System Effector TecA Is a Virulence Factor in Mouse Models of Lung Infection

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex (Bcc), a group of bacteria with members responsible for causing lung infections in cystic fibrosis (CF) patients. The most severe outcome of Bcc infection in CF patients is cepacia syndrome, a disease characterized by necrotizing pneumonia with bacteremia and sepsis. B. cenocepacia is strongly associated with cepacia syndrome, making it one of the most virulent members of the Bcc. Mechanisms underlying the pathogenesis of B. cenocepacia in lung infections and cepacia syndrome remain to be uncovered. B. cenocepacia is primarily an intracellular pathogen and encodes the type VI secretion system (T6SS) effector TecA, which is translocated into host phagocytes. TecA is a deamidase that inactivates multiple Rho GTPases, including RhoA. Inactivation of RhoA by TecA triggers assembly of the pyrin inflammasome, leading to secretion of proinflammatory cytokines, such as interleukin-1b, from macrophages. Previous work with the B. cenocepacia clinical isolate J2315 showed that TecA increases immunopathology during acute lung infection in C57BL/6 mice and suggested that this effector acts as a virulence factor by triggering assembly of the pyrin inflammasome. Here, we extend these results using a second B. cenocepacia clinical isolate, AU1054, to demonstrate that TecA exacerbates weight loss and lethality during lung infection in C57BL/6 mice and mice engineered to have a CF genotype. Unexpectedly, pyrin was dispensable for TecA virulence activity in both mouse infection models. Our findings establish that TecA is a B. cenocepacia virulence factor that exacerbates lung inflammation, weight loss, and lethality in mouse infection models. IMPORTANCE B. cenocepacia is often considered the most virulent species in the Bcc because of its close association with cepacia syndrome in addition to its capacity to cause chronic lung infections in CF patients (1). Prior to the current study, virulence factors of B. cenocepacia important for causing lethal disease had not been identified in a CF animal model of lung infection. Results of this study describe a CF mouse model and its use in demonstrating that the T6SS effector TecA of B. cenocepacia exacerbates inflammatory cell recruitment and weight loss and is required for lethality and, thus, acts as a key virulence factor during lung infection. This model will be important in further studies to better understand TecA’s role as a virulence factor and in investigating ways to prevent or treat B. cenocepacia infections in CF patients. Additionally, TecA may be the founding member of a family of virulence factors in opportunistic pathogens

Reducing Uncertainties in a Wind-Tunnel Experiment using Bayesian Updating

We perform a fully stochastic analysis of an experiment in aerodynamics. Given estimated uncertainties on the principle input parameters of the experiment, including uncertainties on the shape of the model, we apply uncertainty propagation methods to a suitable CFD model of the experimental setup. Thereby we predict the stochastic response of the measurements due to the experimental uncertainties. To reduce the variance of these uncertainties a Bayesian updating technique is employed in which the uncertain parameters are treated as calibration parameters, with priors taken as the original uncertainty estimates. Imprecise measurements of aerodynamic forces are used as observational data. Motivation and a concrete application come from a wind-tunnel experiment whose parameters and model geometry have substantial uncertainty. In this case the uncertainty was a consequence of a poorly constructed model in the pre-measurement phase. These methodological uncertainties lead to substantial uncertainties in the measurement of forces. Imprecise geometry measurements from multiple sources are used to create an improved stochastic model of the geometry. Calibration against lift and moment data then gives us estimates of the remaining parameters. The effectiveness of the procedure is demonstrated by prediction of drag with uncertainty

Panels of chemically-modified heparin polysaccharides and natural heparan sulfate saccharides exhibit differences in binding to Slit and Robo, as well as variation between protein binding and cellular activity.

Heparin/ heparan sulfate (HS) glycosaminoglycans are required for Slit-Robo cellular responses. Evidence exists for interactions between each combination of Slit, Robo and heparin/HS and for formation of a ternary complex. Heparin/HS are complex mixtures displaying extensive structural diversity. The relevance of this diversity has been studied to a limited extent using a few select chemically-modified heparins as models of HS diversity. Here we extend these studies by parallel screening of structurally diverse panels of eight chemically-modified heparin polysaccharides and numerous natural HS oligosaccharide chromatographic fractions for binding to both Drosophila Slit and Robo N-terminal domains and for activation of a chick retina axon response to the Slit fragment. Both the polysaccharides and oligosaccharide fractions displayed variability in binding and cellular activity that could not be attributed solely to increasing sulfation, extending evidence for the importance of structural diversity to natural HS as well as model modified heparins. They also displayed differences in their interactions with Slit compared to Robo, with Robo preferring compounds with higher sulfation. Furthermore, the patterns of cellular activity across compounds were different to those for binding to each protein, suggesting that biological outcomes are selectively determined in a subtle manner that does not simply reflect the sum of the separate interactions of heparin/HS with Slit and Robo

Mutations in Complement Factor H Impair Alternative Pathway Regulation on Mouse Glomerular Endothelial Cells in Vitro

Complement factor H (FH) inhibits complement activation and interacts with glomerular endothelium via its complement control protein domains 19 and 20, which also recognize heparan sulfate (HS). Abnormalities in FH are associated with the renal diseases atypical hemolytic uremic syndrome and dense deposit disease and the ocular disease age-related macular degeneration. Although FH systemically controls complement activation, clinical phenotypes selectively manifest in kidneys and eyes, suggesting the presence of tissue-specific determinants of disease development. Recent results imply the importance of tissue-specifically expressed, sulfated glycosaminoglycans (GAGs), like HS, in determining FH binding to and activity on host tissues. Therefore, we investigated which GAGs mediate human FH and recombinant human FH complement control proteins domains 19 and 20 (FH19-20) binding to mouse glomerular endothelial cells (mGEnCs) in ELISA. Furthermore, we evaluated the functional defects of FH19-20 mutants during complement activation by measuring C3b deposition on mGEnCs using flow cytometry. FH and FH19-20 bound dose-dependently to mGEnCs and TNF- treatment increased binding of both proteins, whereas heparinase digestion and competition with heparin/HS inhibited binding. Furthermore, 2-O-, and 6-O-, but not N-desulfation of heparin, significantly increased the inhibitory effect on FH19-20 binding to mGEnCs. Compared with wild type FH19-20, atypical hemolytic uremic syndrome-associated mutants were less able to compete with FH in normal human serum during complement activation on mGEnCs, confirming their potential glomerular pathogenicity. In conclusion, our study shows that FH and FH19-20 binding to glomerular endothelial cells is differentially mediated by HS but not other GAGs. Furthermore, we describe a novel, patient serum-independent competition assay for pathogenicity screening of FH19-20 mutants.Peer reviewe

Temperature as an indicator of flow in fractured rocks near Oracle, Arizona

Knowledge of flow patterns in fractured rocks is important in understanding hydrology and contaminant transport. Methods of locating flow zones based on geophysical measurements are unreliable because of a lack of correlation between fracture density and hydraulic conductivity. In this work, analysis of data from natural temperature profiles, longterm heating tests, and three-hole recirculation thermal tracer experiments in saturated fractured granite indicates that temperature measurements can be used to identify local flow paths and flow directions in fractured systems. However, adequate controls on background noise and sufficient supplementary information must be incorporated into the testing procedure. Although thermal tracer tests at the Oracle site were inconclusive due to insufficient heat input, the use of heat as a tracer to define larger-scale flow patterns is found to be potentially useful in situations where ample supplies of warmer or cooler water are available.hydrology collectio

River Flow Impacts Cyanobacterial Growth

Biochemistry and Molecular Biology at Wells CollegeCyanobacterial blooms of blue-green algae plague the lakes near Menomonie each summer. I was awarded the opportunity to come to Wisconsin to participate in research to combat this problem as part of the LAKES REU program. My research project primarily looked at the impact of the Red Cedar and Hay Rivers on Tainter Lake and the development of cyanobacterial blooms. I found that as river flow increases there is also an increase in total phosphorus to the lake as well as soluble reactive phosphorus, the type that is most readily available for cyanobacteria to use to grow. While all phosphorus causes blooms, soluble reactive phosphorus should be a target for remediation strategies. So how does soluble reactive phosphorus increase with river flow? To understand the answer you have to look at what is going on physically within the river when a major precipitation event occurs. Most of the time the rivers are in a low flow state, and little phosphorus enters the system. When a storm hits hard and water begins to run off the surface of the surrounding land, the river’s volume increases greatly. Impenetrable surfaces such as tilled farm fields and paved areas are particularly susceptible to surface runoff. This is how major storms that occur throughout the summer months can cause enormous amounts of phosphorus to enter the lake. Storms make the water travel quickly and even violently, including the water at the bottom of the river. As the river volume increases the streambed churns causing phosphorus to re-suspend and dissolve into the water. This increase in soluble reactive phosphorus increases the algae “food” and prepares the next round of algae to multiply. Later in summer months this build-up of phosphorus and algae becomes too great to manage by occasional rain and the smelly, green lake is there to stay until cooler weather comes. So how do we best approach this problem? My findings suggest that the best way to reduce soluble phosphorus in the rivers and lakes is to better manage land in the Red Cedar Watershed. Best management practices for farmlands, as well as dam and lake management, will be key areas to explore for ways to reduce phosphorus from the watershed’s system of streams, rivers and lakes. The goal to ultimately reduce, delay, or eliminate the algal bloom is widely desired, but all people will have to work together to meet this goal. By gaining a better understanding of the problem, mitigation strategies can be tailored to best meet the needs of the Red Cedar Watershed and Tainter and Menomin Lakes

Complement regulation by heparan sulfate and factor H on the endothelial glycocalyx

Contains fulltext : 201848.pdf (publisher's version ) (Open Access)Radboud University, 16 april 2019Promotor : Berden, J.H.M. Co-promotor : Vlag, J. van de

A summary and discussion of hydrologic data from the Calico Hills nonwelded hydrogeologic unit at Yucca Mountain, Nevada

This report is a summary of available hydrologic data from in situ and laboratory testing of the Calico Hills nonwelded hydrogeologic unit, including hydraulic conductivity, porosity, saturation, pore-size distribution and parameters from curve-fits to pressure-saturation data. Sample statistics of hydraulic conductivity, porosity and saturation data for vitric, devitrified and zeolitic tuffs are presented and discussed. While a high degree of variability is observed in both laboratory and in situ hydraulic conductivity measurements, uncertainties arising from differences in size of laboratory test samples, sample handling, test procedures and insufficient number of samples point to the need for additional data of specific types to adequately characterize the unit. Hydrologic issues related to transport analysis in the Calico Hills nonwelded hydrogeologic unit at Yucca Mountain are discussed together with recommendations for future work. The compiled data are included as an appendix

Complement regulation by heparan sulfate and factor H on the endothelial glycocalyx

Contains fulltext : 201848.pdf (publisher's version ) (Open Access)Radboud University, 16 april 2019Promotor : Berden, J.H.M. Co-promotor : Vlag, J. van de