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

Development of Microfluidic Cell Culture Models of the Blood-Retinal Barriers

Tohoku University梶弘和課

Contribution of stromal cells to the formation and stabilisation of blood microvessels

Vasculogenesis, the creation of new blood vessels, occurs due to an inherent ability of endothelial cells (ECs) to self-assemble and form tubules. Additionally, many studies have shown that stromal cells, connective tissue cells of any organ, including fibroblasts (FBs), play a vital role in the formation and stabilisation of new vessels, mostly chemically by producing and secreting growth factors. However, still we lack ground understanding of the mechanical contribution of FBs to vasculogenesis. In this study, employing microfluidic platforms, we aimed to address the mechanical role of FBs in vascularisation. Applying a 7-channel microfluidic platform, we encapsulated ECs with 3 different conditions: mono-cultured (MC, embedding only ECs within a hydrogel), paracrine co-cultured (PCC, embedding ECs and FBs separately in two hydrogels) and juxtacrine co-cultured (JCC, embedding ECs mixed with FBs in a hydrogel) to investigate the impact of FB presence on formation of functional microvessels. Imaging techniques and dextran perfusion revealed that the cluster-like structures formed in the device seeded with MC and PCC conditions were not functional in terms of perfusability and permeability and deteriorated within a week while the microvessels developed in the device seeded with JCC condition were functional, well-interconnected and survived much longer (~3 weeks), indicating that the direct physical interaction between FBs and ECs is crucial for the formation of functional blood microvessels. Also, chemical perturbation of mechanotransduction genes, YAP, Src, Wnt/β-catenin, RhoA, and FAK, in both ECs and FBs, resulted in either the inhibition of microvessel formation or the development of microvessels which were significantly different in morphology, perfusability, vessel length, diameter, and coverage area compared to control microvessels, demonstrating that mechanotransduction pathways play key roles in vascularisation. Additionally, using siRNA approach, we inhibited the same genes only in FBs to examine the mechanical contribution of FBs to vascularisation. This revealed that ECs co-cultured with siRNA-inhibited FBs (excluding RhoA) retained their ability to form microvessels. However, further characterisations demonstrated that the microvessels did not resemble control microvessels in terms of permeability, perfusability, tissue stiffness, barrier function, morphology, and vessel topology. Together, these results highlight the mechanical contribution of FBs to the formation, morphogenesis and function of microvessels and suggests that FBs are intrinsic mechanical promoters and stabilisers of microvessels. Such knowledge on the mechanisms underlying the vascularisation, will be useful in further developing vascularisation strategies for organ-specific, disease-specific, and cancer-specific tissue engineering and regenerative medicine applications

In vitro models of biological barriers for nanomedical research

Nanoconstructs developed for biomedical purposes must overcome diverse biological barriers before reaching the target where playing their therapeutic or diagnostic function. In vivo models are very complex and unsuitable to distinguish the roles plaid by the multiple biological barriers on nanoparticle biodistribution and effect; in addition, they are costly, time-consuming and subject to strict ethical regulation. For these reasons, simplified in vitro models are preferred, at least for the earlier phases of the nanoconstruct development. Many in vitro models have therefore been set up. Each model has its own pros and cons: conventional 2D cell cultures are simple and cost-effective, but the information remains limited to single cells; cell monolayers allow the formation of cell-cell junctions and the assessment of nanoparticle translocation across structured barriers but they lack three-dimensionality; 3D cell culture systems are more appropriate to test in vitro nanoparticle biodistribution but they are static; finally, bioreactors and microfluidic devices can mimicking the physiological flow occurring in vivo thus providing in vitro biological barrier models suitable to reliably assess nanoparticles relocation. In this evolving context, the present review provides an overview of the most representative and performing in vitro models of biological barriers set up for nanomedical research

Green Synthesis of Silver Nanoparticles using Achillea biebersteinii Flower Extract and Its Anti-Angiogenic Properties in the Rat Aortic Ring Model

Silver nanoparticles display unique physical and biological properties which have attracted intensive research interest because of their important medical applications. In this study silver nanoparticles (Ab.Ag-NPs) were synthesized for biomedical applications using a completely green biosynthetic method using Achillea biebersteinii flowers extract. The structure and properties of Ab.Ag-NPs were investigated using UV-visible spectroscopic techniques, transmission electron microscopy (TEM), zeta potential and energy dispersive X-ray spectrometers (EDS). The UV-visible spectroscopic analysis showed the absorbance peak at 460 nm, which indicates the synthesis of silver nanoparticles. The average particle diameter as determined by TEM was found to be 12 ± 2 nm. The zeta potential analysis indicated that Ab.Ag-NPs have good stability EDX analysis also exhibits presentation of silver element. As angiogenesis is an important phenomenon and as growth factors imbalance in this process causes the acceleration of several diseases including cancer, the anti-angiogenic properties of Ab.Ag-NPs were evaluated using the rat aortic ring model. The results showed that Ab.Ag-NPs (200 μg/mL) lead to a 50% reduction in the length and number of vessel-like structures. The synthesized silver nanoparticles from the Achillea biebersteinii flowers extract, which do not involve any harmful chemicals were well-dispersed and stabilized through this green method and showed potential therapeutic benefits against angiogenesis

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

96 perfusable blood vessels to study vascular permeability in vitro

Analytical BioScience

Transendothelial Movement of Adiponectin in Diabetic Vasculature

Adiponectin is one of the most abundant circulatory hormone that plays an important role on homeostasis of glucose and lipid, oxidative stress, and inflammation by enhancing insulin sensitivity. It is highly implicated to pathogenesis of metabolic syndrome. This thesis examined: (study 1) glucocorticoids effect on adiponectin flux by regulation of permeability and its mechanism involved, (study 2) impact of high glucose on transendothelial movement of adiponectin and a whole-body biodistribution to understand functional significance, (study3) influence of iron overload on endothelial permeability of adiponectin to investigate the regulatory mechanism. Findings from study 1 indicated that glucocorticoids altered tight junction profiles that led to reduce endothelial paracellular permeability and to decrease adiponectin contents in rat skeletal muscle. In study 2, the data demonstrated that hyperglycemia decreased vascular permeability and resulted in increased adiponectin transendothelial movement, which observations were tested by multifaceted vasculature platforms in vivo, ex vivo and 2D & 3D in vitro with high glucose treatment. Lastly, study 3 showed that iron overload induced oxidative stress and altered tight junction expression to elevate permeability of endothelial monolayers. This increased adiponectin movement across the endothelial barrier. In summary, my studies demonstrated that adiponectin transendothelial movement was regulated by vascular permeability. The alteration of permeability relied on expression of tight junction and its regulatory mechanism resulted from diabetic conditions

Roles of nitric oxide and shear stress in the regulation of microvessel permeability in intact rat mesenteric venules

Mechanical forces have been indicated to play important roles in the regulation of inflammatory cell interaction with endothelium resulting in localized leakage formation and contributing to many disease-associated microvascular dysfunctions. However, most of the mechanical force related studies were conducted in vitro. The underlying mechanisms are still controversial. There is a need to investigate how shear stress regulates the endothelial cell (EC) signaling and related vascular barrier function using intact microvessels with experimental conditions closely replicating in vivo situations. The overall aim of my dissertation is to understand the molecular and cellular mechanisms of how shear stress and nitric oxide (NO) regulate microvessel function under physiological and pathological conditions. Studies were conducted on individually perfused intact rat mesenteric venules.;It is well known that shear stress is one of most important regulators in stimulating endothelial cells to produce NO. NO, in addition to being a potent vasodilator, has also been considered a double edged sword -mediator in inflammation. Under basal conditions, it prevents leukocyte and platelet adhesion, whereas under inflammatory conditions, the inflammatory mediator-induced excessive NO production contributes to permeability increases. In Chapter 2, we investigated the roles of endothelial basal NO production in leukocyte adhesion and adhesion-induced changes in microvessel permeability. The results indicated that the application of the eNOS specific inhibitor, caveolin-1 scaffolding peptide (CAV), caused reduction of basal NO and promoted ICAM-1-mediated leukocyte adhesion through Src activation-mediated ICAM-1 phosphorylation. Also, CAV-induced leukocyte adhesion was uncoupled from leukocyte oxidative burst and microvessel barrier function, unless in the presence of a secondary stimulation.;In Chapter 3, we investigated the roles of shear stress (SS) in the regulation of microvessel permeability and its related EC signaling involving blood cells in individually perfused intact microvessels. Our results demonstrated that in response to a sudden change of SS, transient shear magnitude-dependent increases in EC [Ca2+]i occurred only in vessels perfused with whole blood or perfusate containing RBCs, which was correlated with EC gap formation illustrated by fluorescent microsphere accumulation. Carbenoxolone, a Pannexin 1 inhibitor, significantly reduced shear magnitude-dependent ATP release from RBCs and also abolished SS-induced increases in EC [Ca 2+]i and EC gap formation. Meanwhile, both plasma and whole blood perfusion induced shear magnitude-dependent NO production and eNOS-Ser 1177 phosphorylation.;It is unknown how EC sense SS, but the Glycocalyx (GCX), a layer of proteoglycans covering the endothelium, has been implicated as a mechanical sensor for changes in SS in vitro. The objective of chapter 4 is to identify the changes in GCX in microvessels of streptozotocin-induced diabetic rats and evaluate the associated changes in sensing SS and SS-induced NO production in individually perfused venules of diabetic rats. Our results indicated that the impaired GCX in diabetic microvessels enhances EC response to mechanical force and potentiates NO production and EC responses to ATP, resulting in enhanced endothelial gap formation.;Advances in micromanufacturing and microfluidic technologies have enabled a variety of insights into biomedical sciences while curtailing the high experimental costs and complexities associated with animals and in vivo studies. In Chapter 5, we presented and discussed our research work in creating engineered microvessels using a microfluidic platform and demonstrated the formation of the microvascular network in vitro and validated the key features that have been observed in microvessels in vivo. In our future studies, this may provide us a novel platform for studying spatial and temporal change of shear stress in the regulation of microvessel function in a close in vivo situation.;In conclusion, we revealed the role of shear stress and NO in the regulation of endothelial cell signaling and microvessel permeability in vivo, involving blood and non-blood components. The results also suggest the potential in using a microfluidic device in studying the physiological microvessel function