A Novel Microbial Source Tracking DNA Microarray Used for Pathogen Detection in Environmental Systems

Pathogen detection and the identification of fecal contamination sources can be challenging in environmental and engineered treatment systems. Factors including pathogen diversity and ubiquity of fecal indicator bacteria hamper risk assessment and remediation of contamination sources. Therefore, a quick method that can detect and identify waterborne pathogens in environmental systems is needed. In this work, a custom microarray targeting pathogens (viruses, bacteria, protozoa), microbial source tracking (MST) markers, mitochondria DNA (mtDNA) and antibiotic resistance genes was used to detect over 430 selected gene targets in whole genome amplification (WGA) DNA and complementary DNA (cDNA) isolated from sewage and animal (avian, cattle, poultry and swine) feces, freshwater and marine water samples, sewage spiked surface water samples, treated wastewater and sewage contaminated produce.;A combination of perfect match and mismatch probes on the microarray reduced the likelihood of false positive detections, thus increasing the specificity of the microarray for various gene targets. A linear decrease in fluorescence of positive probes over a 1:10 dilution series demonstrated a semi-quantitative relationship between gene concentrations in a sample and microarray fluorescence. Various pathogens, including norovirus, Campylobacter fetus, Helicobacter pylori, Salmonella enterica, and Giardia lamblia were detected in sewage via the microarray, as well as MST markers and resistance genes to aminoglycosides, beta-lactams, and tetracycline. Sensitivity (percentage true positives) of MST results in sewage and animal waste samples (21--33%) was lower than specificity (83--90%, percentage of true negatives). Next generation sequencing (NGS) of DNA from the fecal samples revealed two dominant bacterial families that were common to all sample types: Ruminococcaceae and Lachnospiraceae. Five dominant phyla and 15 dominant families comprised 97% and 74%, respectively, of sequences from all fecal sources.;Waterborne pathogens were also detectable via the microarray in freshwater, marine water and sewage spiked surface water samples as well as treated wastewater. Ultrafiltration was used to concentrate microorganisms (bacteria, viruses, protozoa and parasites) from several liters of environmental and treated water samples. Dead-end ultrafiltration (DEUF) was shown to have a 61.4 +/- 47.8 % recovery efficiency and 46-fold concentration increasing ability. Then WGA was utilized to increase gene copies and lower the microarray detection limit. Viruses, including adenovirus, bocavirus, Hepatitis A virus, and polyomavirus were detected in human associated water samples as well as pathogens like Legionella pneumophila, Shigella flexneri, C. fetus and genes coding for resistance to aminoglycosides, beta-lactams, tetracycline. Microbial source tracking results indicate that sewage spiked freshwater and marine samples clustered separately from other fecal sources including wild and domestic animals via non-metric dimensional scaling. A linear relationship between qPCR and microarray fluorescence was found, indicating the semi-quantitative nature of the MST microarray.;Multiple displacement amplification (MDA), which is an important type of WGA, is a widely used tool to amplify genomic nucleic acids. The strong amplification efficiency of MDA and low initial template requirement make MDA an attractive method for environmental molecular and NGS studies. However, like other nucleic acid amplification techniques, various factors may influence MDA efficiency including template concentration (e.g. rare species swamping out), GC amplification bias and genome length favoring amplification of longer genomes. It was found that MDA increased nucleic acids in mixed environmental samples approximately 4.24 +/- 1.40 (log, average +/- standard deviation) for 16S rRNA gene of Enterococcus faecalis, 1.90 +/- 1.70 for RNA polymerase gene of human norovirus, 8.83 +/- 2.88 for T antigen gene of human polyomavirus, 3.83 +/- 0.93 for uidA gene of Escherichia coli, 4.96 +/- 0.32 for invA gene of S. enterica and 8.77 +/- 2.85 for 16S rRNA gene of human Bacteroidales. The template length, concentration and GC content were found to influence MDA efficiency. The results mainly show that the MDA will be more efficient the longer the template length, the greater the initial concentration of nucleic acids and the lower the GC content of the template.;Overall, the results of this work show that 1) the microarray and sample handling technique is suitable for pathogen detection from feces and sewage; 2) when combined with ultrafiltration techniques, the microarray can also be used as a pathogen detection tool in environmental waters; 3) template length, and initial concentration increase MDA efficiency, but higher GC content template negatively effects MDA efficiency. The proposed microarray can be used for pathogen detection in feces, wastewater treatment plant sewage, treated wastewater and environmental waters. Further the proposed method is potentially applicable to pathogen/microorganism detections on vegetables, seafood, in hospital settings, industrial wastewater, and aquaculture settings

The Role of Wall Shear Stress in the Failure of Arteriovenous Fistula for Hemodialysis Vascular Access

Native arteriovenous fistulas created in the forearm between the radial artery and the cephalic vein are currently considered the best vascular access for hemodialysis in terms of outcome and safety. However, this type of access is associated with a significant failure rate that is generally caused by intimal hyperplasia development and consequent stenosis. Computational studies have shown that intimal hyperplasia is preferentially localized in regions of the fistula that are exposed to disturbed wall shear stresses. This thesis reports the development and testing of a novel, real-time controlled cone-and-plate device to assess the role of changes in shear stress induced by fistula creation on the activation state of endothelial cells and paracrine signalling in vitro. The selected shear stress waveforms were representative of the pulsatile/unidirectional waveforms in zones that are generally not affected by hyperplasia or the disturbed/reciprocating waveforms present in the high-risk stenosis areas. Experimental results showed that after 48 hours of flow exposure, these stimuli elicited differential effects on human umbilical vein endothelial cells monolayers. Pulsatile waveforms induced major cellular shape remodeling and upregulation of the transcription factor KLF2 mRNA expression. On the contrary cells exposed to reciprocating flows showed a lack of cytoskeletal remodeling, upregulation of the enzyme PLDl , the integrin subunit ITGA4 and RASA1 (coding for the protein activator p120RasGAP) mRNA expression and an increased production of chemoattractant cytokines MCP-1 and IL-8. Furthermore, it was observed that human smooth muscle cells incubated for 24 hours with medium conditioned by endothelial cells exposed to reciprocating shear stress stimuli, had an increased rate of proliferation compared to smooth muscle cells cultured in medium conditioned by endothelial cells exposed to pulsatile waveforms. These results confirm potential links between disturbed wall shear stress and arteriovenous fistula failure

A practical review on the measurement tools for cellular adhesion force

Cell cell and cell matrix adhesions are fundamental in all multicellular organisms. They play a key role in cellular growth, differentiation, pattern formation and migration. Cell-cell adhesion is substantial in the immune response, pathogen host interactions, and tumor development. The success of tissue engineering and stem cell implantations strongly depends on the fine control of live cell adhesion on the surface of natural or biomimetic scaffolds. Therefore, the quantitative and precise measurement of the adhesion strength of living cells is critical, not only in basic research but in modern technologies, too. Several techniques have been developed or are under development to quantify cell adhesion. All of them have their pros and cons, which has to be carefully considered before the experiments and interpretation of the recorded data. Current review provides a guide to choose the appropriate technique to answer a specific biological question or to complete a biomedical test by measuring cell adhesion

Development And Validation Of A Flow Device To Study Platelet Function In Vitro And Elucidating The Role Of Thymosin β 4 In Various Physiological Processes

Parallel plate flow chambers, simulating in vivo fluid shear stress, provide a real time insight into the dynamic process of platelet aggregation and investigation of endothelial cell response to shear stress. This thesis describes the design and validation of - 1) A simple parallel plate flow chamber to study effects of shear stress on endothelial cells. This flow chamber is easy to use, inexpensive and fast to manufacture as compared to the flow devices reported previously. Moreover, it..

Strength, liquefaction and cone penetration test results in unsaturated silty tailings

The behaviour of two silty tailings are explored, especially their propensity for static liquefaction, and how they may be characterised using the cone penetration test (CPT), when unsaturated. The focus is on loose states and high degrees of saturation. Firstly the tailings are characterised, using triaxial tests involving constant suction and constant mass (closed system) conditions to establish stress-strain behaviours, and filter paper and pressure plate tests to establish water retention properties. Consideration of the closed system condition is novel. It is important because an instability commonly develops quickly, and deformation may be rapid, meaning the air and water inside the tailings may not be able to exit. A bounding surface plasticity model is adapted to suit the closed system condition. The triaxial test results are used to calibrate the model, with good agreements achieved. Model simulations are then used to identify which factors influence instability. Additional simulations to mimic rising water tables under constant total stress states in the field are also shown. Results are added to charts which relate peak and post-liquefaction strengths, as well as collapse lines, to measures of initial state, for unsaturated conditions, for use in practice. A number of CPTs are performed on the tailings in a calibration chamber. Interpretations are made, incorporating other CPT results, correlating the measured cone resistances to the tailings’ states for saturated and unsaturated conditions. A cavity expansion analysis is then performed, using the bounding surface model and the similarity technique, novel in its consideration of the closed system condition. Cavity expansion results are compared to the CPT results, a linear proportionality between cavity wall pressure and cone resistance is identified, and charts are produced enabling CPTs to be interpreted in unsaturated tailings whatever the initial state. Two types of behaviour are observed, depending on the volume of air present relative to the overall tailings volume, i.e. v_a/v. Specifically, a closed system condition is relevant to when v_a/v 0.15 the penetration rate is unimportant, as a pseudo drained condition prevails around the cone tip

Applying Chaotic Advection to Rheology: an In Situ Structuring Rheometer

A prototype In Situ Structuring Rheometer (ISSR) was designed and implemented to study changes in shear viscosity of polymer blends and composites while processing them in such a way as to control the evolution of microstructure. The ISSR is based on a regime of fluid mechanics known as chaotic advection, in which simple time-periodic flow fields can cause fluid particles to move chaotically. Chaotic advection is also the basis of Smart Blending, a technology employed to process polymer blends having a variety of morphologies at a fixed composition, and polymer composites in which the additives have been arranged into layered structures or networks. Smart Blending has been implemented as batch devices or continuous flow devices, with a device of the former type providing the basis for the ISSR. Designed as a test cell to be fitted into a commercial instrument so as to leverage its measurement capability, the core of the ISSR is a cylindrical blending cavity the end surfaces of which are formed by rotatable disks which induce stirring. While the upper disk is an attachment for the commercial instrument, the lower disk has an independent drive system. The ISSR also includes a heating chamber, temperature control systems and a purge gas system. Alternate counter-rotation of the disks through an appropriate displacement leads to a chaotic flow. The design of the ISSR and experiments conducted using it were guided by modeling. The result is that even as the microstructure in the sample is being controllably formed, the shear viscosity is measured each time the upper disk rotates. In contrast, conventional rheometry using a parallel-plate or cone-plate test cell involves mixing materials as melts beforehand, with a polymer blend usually having a droplet morphology and a composite usually having the additive randomly dispersed throughout the polymer matrix. Three types of systems were processed and studied using the ISSR. At least three samples of each system were processed to different extents, cryogenically fractured and examined using scanning electron microscopy (SEM). By so doing, the trends in viscosity were related to progressive structure development, which is the controlled evolution and retention of particular blend and composite morphologies, as has been documented in previous chaotic advection blending studies. The first system was a compatible blend of low density polyethylene (LDPE) and high density polyethylene (HDPE), for which the viscosity initially rose and eventually reached a plateau, which was consistent with a model that showed no change in viscosity with the number of layers. Blend samples at different stages of processing showed the initial formation of layers and the development of nanoscale features as these layers refined. The second system was a composite of linear low density polyethylene (LLDPE) and carbon black (CB), for which the shear viscosity slowly decreased with continued processing. Micrographs indicated that the carbon black initially formed coarse striations and may have subsequently formed networks, as was observed in previous studies using related chaotic advection blending devices. The third system, an immiscible blend of LDPE and polypropylene (PP), exhibited a nearly constant viscosity. Repeatability of viscosity data was an issue for all three systems. Several problems with this prototype were identified as potential factors: misalignment of the cavity components, sample leakage, temperature cycling of the sample, and coordination of disk motions. To address these problems, it is recommended that the cavity seal be improved, the temperature control systems studied more thoroughly, and the disk motions coordinated automatically in a future ISSR

Fluid dynamic analysis of flow in orbiting dishes and the effects of flow on shear stress and endothelial cellular responses.

This work presents a novel comprehensive effort to understand the fluid dynamics within orbiting dishes and the effects of the resulting oscillating fluid flow on shear and endothelial cellular responses within the dishes. It is well documented that hemodynamic parameters, especially wall shear stress (WSS), have been shown to play important roles in altering various endothelial cellular responses, intracellular pathways and gene expression, and to have significant impacts on disease progression, such as atherosclerotic plaque development. In practice, wall shear stress (WSS) is oscillatory rather than steady due to the travelling waveform and varies across the surface of the dish at any instant in time. The first part of this effort presents a computational model which provides complete spatial and temporal resolution of oscillatory WSS over the bottom surface of an orbiting Petri dish throughout the orbital cycle. The model was reasonably well validated by the analytical solution and the results were compared to the tangential WSS magnitudes obtained using one-dimensional optical velocimetry at discreet locations on the bottom of an orbiting dish. A thorough fluid dynamic analysis was performed in the next part of this work to understand the fluid motion inside the dish by investigating the system properties that affect WSS. To identify the effects of each of those properties on WSS, a dimensional analysis study was performed which includes analyses of three dimensionless parameters - Slope ratio, Froude Number, and Stokes Number. A fourth Reynolds Number was held constant. By analyzing a range from low to high values for each of the parameters, transition points for each of the flow parameters were determined. Further the nature of WSS at different radii (20%, 40%, 60% and 80% of the maximum radius of the dish) on the bottom surface was studied as a function of combinations of various dimensionless parameters. In the last part of this study, this model was applied to understand the effects of oscillatory WSS on endothelial cellular responses, including cell proliferation, morphology, and atherogenic gene expression. Since WSS on the bottom of the dish is two-dimensional, a new directional oscillatory shear index (DOSI) was developed to quantify the directionality of oscillating shear. DOSI approached zero for bidirectional oscillatory shear of equal magnitudes near the center and approached one for unidirectional oscillatory shear near the wall, where large tangential WSS dominated a much smaller radial component. Cellular responses including cell proliferation, area, shape index, orientation, and atherogenic gene expressions at mRNA level (1-CAMl, E-Selectin, IL-6) were then correlated with different DOS I level under the same flow conditions. A comprehensive statistical analysis demonstrated that DOSI significantly affects all the responses, indicating that, in addition to shear magnitudes, directionality and the oscillatory nature of shear significantly influence cellular responses

Pressure development due to viscous fluid flow through a converging gap

The behaviour of fluid flow in industrial processes is essential for numerous applications and there have been vast amount of work on the hydrodynamic pressure generated due to the flow of viscous fluid. One major manifestation of hydrodynamic pressure application is the wire coating/drawing process, where the wire is pulled through a unit either conical or cylindrical bore filled with a polymer melt that gives rise to the hydrodynamic pressure inside the unit. The hydrodynamic pressure distribution may change during the process due to various factors such as the pulling speed, process temperature, fluid viscosity, and geometrical shape of the unit (die). This work presents the process of designing a new plasto-hydrodynamic pressure die based on a tapered-stepped-parallel bore shape; the device consists of a fixed hollow outer cylinder and an inner rotating shaft, where the hollow cylinder represents a pressure chamber and the rotating shaft represents the moving surface of the wire. The geometrical shape of the bore is provided by different shaped inserts to set various gap ratios, ancl the complex geometry of the gap between the shaft and the pressure chamber is filled with viscous fluid materials. The device allows the possibility of determining changes in the hydrodynamic pressure as the shaft speed is altered while different fluid viscosity during the process is considered. A number of experimental procedures and methods have been carried out to determine the effects of various shaft speeds by using Glycerine at 1 to 18 °C and two different types of silicone oil fluids at 1 to 25 °C on the hydrodynamic pressure and shear rate. Viscosities of the viscous fluids were obtained at atmospheric pressure by using a Cone-plate Brookfield viscometer at low shear rate ranges. Moreover, Computational fluid dynamics (CFD) was used to develop and analyze computational simulation models that demonstrate the pressure units, which studies the drawing process involving viscous fluids in a rotating system. A finite volume technique was used to observe the change in fluid viscosity during the process based on non-Newtonian characteristics at high shear rate ranges. The maximum shaft speed used in these models was 1.5m.sec'1. Results from experimental and Computational models were presented graphically and discussed

Hydrodynamic Regulation of Monocyte Inflammatory Response to an Intracellular Pathogen

Systemic bacterial infections elicit inflammatory response that promotes acute or chronic complications such as sepsis, arthritis or atherosclerosis. Of interest, cells in circulation experience hydrodynamic shear forces, which have been shown to be a potent regulator of cellular function in the vasculature and play an important role in maintaining tissue homeostasis. In this study, we have examined the effect of shear forces due to blood flow in modulating the inflammatory response of cells to infection. Using an in vitro model, we analyzed the effects of physiological levels of shear stress on the inflammatory response of monocytes infected with chlamydia, an intracellular pathogen which causes bronchitis and is implicated in the development of atherosclerosis. We found that chlamydial infection alters the morphology of monocytes and trigger the release of pro-inflammatory cytokines TNF-α, IL-8, IL-1β and IL-6. We also found that the exposure of chlamydia-infected monocytes to short durations of arterial shear stress significantly enhances the secretion of cytokines in a time-dependent manner and the expression of surface adhesion molecule ICAM-1. As a functional consequence, infection and shear stress increased monocyte adhesion to endothelial cells under flow and in the activation and aggregation of platelets. Overall, our study demonstrates that shear stress enhances the inflammatory response of monocytes to infection, suggesting that mechanical forces may contribute to disease pathophysiology. These results provide a novel perspective on our understanding of systemic infection and inflammation