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 key role of nitric oxide in hypoxia: hypoxic vasodilation and energy supply-demand matching

Significance: a mismatch between energy supply and demand induces tissue hypoxia with the potential to cause cell death and organ failure. Whenever arterial oxygen concentration is reduced, increases in blood flow - 'hypoxic vasodilation' - occur in an attempt to restore oxygen supply. Nitric oxide is a major signalling and effector molecule mediating the body's response to hypoxia, given its unique characteristics of vasodilation (improving blood flow and oxygen supply) and modulation of energetic metabolism (reducing oxygen consumption and promoting utilization of alternative pathways). Recent advances: this review covers the role of oxygen in metabolism and responses to hypoxia, the hemodynamic and metabolic effects of nitric oxide, and mechanisms underlying the involvement of nitric oxide in hypoxic vasodilation. Recent insights into nitric oxide metabolism will be discussed, including the role for dietary intake of nitrate, endogenous nitrite reductases, and release of nitric oxide from storage pools. The processes through which nitric oxide levels are elevated during hypoxia are presented, namely (i) increased synthesis from nitric oxide synthases, increased reduction of nitrite to nitric oxide by heme- or pterin-based enzymes and increased release from nitric oxide stores, and (ii) reduced deactivation by mitochondrial cytochrome c oxidase. Critical issues: several reviews covered modulation of energetic metabolism by nitric oxide, while here we highlight the crucial role NO plays in achieving cardiocirculatory homeostasis during acute hypoxia through both vasodilation and metabolic suppression Future directions: we identify a key position for nitric oxide in the body's adaptation to an acute energy supply-demand mismatc

Whole-body heat stress and exercise stimulate the appearance of platelet microvesicles in plasma with limited influence of vascular shear stress

Intense, large muscle mass exercise increases circulating microvesicles, but our understanding of microvesicle dynamics and mechanisms inducing their release remains limited. However, increased vascular shear stress is generally thought to be involved. Here, we manipulated exercise-independent and exercise-dependent shear stress using systemic heat stress with localized single-leg cooling (low shear) followed by single-leg knee extensor exercise with the cooled or heated leg (Study 1, n = 8) and whole-body passive heat stress followed by cycling (Study 2, n = 8). We quantified femoral artery shear rates (SRs) and arterial and venous platelet microvesicles (PMV-CD41+) and endothelial microvesicles (EMV-CD62E+). In Study 1, mild passive heat stress while one leg remained cooled did not affect [microvesicle] (P ≥ 0.05). Single-leg knee extensor exercise increased active leg SRs by ~12-fold and increased arterial and venous [PMVs] by two- to threefold, even in the nonexercising contralateral leg (P < 0.05). In Study 2, moderate whole-body passive heat stress increased arterial [PMV] compared with baseline (mean±SE, from 19.9 ± 1.5 to 35.5 ± 5.4 PMV.μL-1.103, P < 0.05), and cycling with heat stress increased [PMV] further in the venous circulation (from 27.5 ± 2.2 at baseline to 57.5 ± 7.2 PMV.μL-1.103 during cycling with heat stress, P < 0.05), with a tendency for increased appearance of PMV across exercising limbs. Taken together, these findings demonstrate that whole-body heat stress may increase arterial [PMV], and intense exercise engaging either large or small muscle mass promote PMV formation locally and systemically, with no influence upon [EMV]. Local shear stress, however, does not appear to be the major stimulus modulating PMV formation in healthy humans

Exercise-Derived Microvesicles: A Review of the Literature

Initially suggested as simple cell debris, cell-derived microvesicles (MVs) have now gained acceptance as recognized players in cellular communication and physiology. Shed by most, and perhaps all, human cells, these tiny lipid-membrane vesicles carry bioactive agents, such as proteins, lipids and microRNA from their cell source, and are produced under orchestrated events in response to a myriad of stimuli. Physical exercise introduces systemic physiological challenges capable of acutely disrupting cell homeostasis and stimulating the release of MVs into the circulation. The novel and promising field of exercise-derived MVs is expanding quickly, and the following work provides a review of the influence of exercise on circulating MVs, considering both acute and chronic aspects of exercise and training. Potential effects of the MV response to exercise are highlighted and future directions suggested as exercise and sports sciences extend the realm of extracellular vesicles

Experimentation, modeling and control of calcium dynamics in human vascular endothelial cells

Ph.DDOCTOR OF PHILOSOPH

The Interplay between Chemistry and Mechanics in the Transduction of a Mechanical Signal into a Biochemical Function

There are many processes in biology in which mechanical forces are generated. Force-bearing networks can transduce locally developed mechanical signals very extensively over different parts of the cell or tissues. In this article we conduct an overview of this kind of mechanical transduction, focusing in particular on the multiple layers of complexity displayed by the mechanisms that control and trigger the conversion of a mechanical signal into a biochemical function. Single molecule methodologies, through their capability to introduce the force in studies of biological processes in which mechanical stresses are developed, are unveiling subtle intertwining mechanisms between chemistry and mechanics and in particular are revealing how chemistry can control mechanics. The possibility that chemistry interplays with mechanics should be always considered in biochemical studies.Comment: 50 pages, 18 figure

The Roles of Primary Cilia in Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited genetic disorder that results in progressive renal cyst formation with ultimate loss of renal function and other systemic disorders. These systemic disorders include abnormalities in cardiovascular, portal, pancreatic and gastrointestinal systems. ADPKD is considered to be among the ciliopathy diseases due to the association with abnormal primary cilia function. In order to understand the full course of primary cilia and its association with ADPKD, the structure, functions and role of primary cilia have been meticulously investigated. As a result, the focus on primary cilia has emerged to support the vital roles of primary cilia in ADPKD. The primary cilia have been shown to have not only a mechanosensory function but also a chemosensory function. Both structural and functional defects in primary cilia result in cystic kidney disease and vascular hypertension. Thus, the mechanosenory and chemosensory functions will be analyzed in regards to ADPKD

The Dynamic Roles of Red Blood Cell in Microcirculation

Erythrocytes (otherwise known as red blood cells (RBCs)), are the most common cell type in the body. They are responsible for oxygen (O2) transportation as well as carbon dioxide (CO2) exchange. Different from most cells, red cells have no nuclei in mammals due to the enucleation during the maturation. The structure of erythrocytes was shown to have a phospholipid bilayer membrane, membrane proteins and cell skeleton. It provides the stability that RBCs need for the circulation in the body systems. Also, this well-established structure makes it possible for them to accomplish ion and gas exchange, which therefore keeps the osmolality and pressure stable for extracellular and intracellular environment. Although a great variety of red cell characteristics have been investigated, the mechanism and kinetics of RBCs under certain environmental stimulation have not been well studied. In this work, we studied the development of cell membrane by testing the deformability change of erythrocytes during maturation. With the design of our microfluidic channels in ex vivo experiments, we then learned that RBC can work not only as O2 transporter but also as oxygen sensor itself. When oxygen level decrease, TBC membrane becomes softer and leads to blood flow increase eventually. We then investigated the mechanism of RBC membrane change on a molecular level to study the mechanism of RBC deformability change under hypoxia. We matched our findings in both in vivo and ex vivo experiments. Via in vivo experiments, we could even connect cerebral circulation to neuroactivity. Furthermore, the behavior of RBCs under hypoxia and in shear flow, such as the ATP release, was studied as well via ex vivo experiments. In the study, we focused on the mechanosensitive channel Piezo1 on RBC membrane and found the connection between this ion channel and RBC ATP release

Investigating the Role of Mechanosensitive Ion Channels in Urothelial Cell Pressure Mechanotransduction

Overactive bladder (OAB) is a bladder disorder that is characterized by bladder storage symptoms of urgency with or without urge incontinence, frequency, nocturia, and, as of 2003, affected approximately 16.5% of adults in the United States with an annual treatment cost of over $65 billion. While therapies are available to mitigate the symptoms of OAB, there are no treatments for the cause of OAB, due to the lack of understanding of the etiology of the disorder. Recent research has provided evidence that the bladder urothelium is not just a passive barrier, but is also sensitive to various chemical and mechanical stimuli and responds by releasing neurotransmitters (NO, ATP) to participate in the sensory function of the bladder. Thus, it has been suggested that overactive bladder is caused, at least in part, by altered sensitivity to bladder fullness by urothelial cells (UCs). While previous research has demonstrated that the urothelium responds to tissue stretch induced by elevated pressure, the role of hydrostatic pressure alone in the cellular events was unclear. Therefore, this doctoral thesis sought to decouple the two stimuli and investigate the effects of pressure on UCs, especially the role of select ion channels in UC pressure mechanotransduction. Overall, the objective of this project was to test the hypothesis that UCs sense hydrostatic pressure via activation of membrane-bound ion channels, and that these cells respond by releasing ATP. In order to test this hypothesis, multiple in vitro studies were performed in which UCs were subjected to controlled hydrostatic pressure while monitoring their response to the stimulus. First, using a custom-made pressure chamber, rat bladder UCs were exposed to sustained hydrostatic pressure (5-20 cmH2O) for up to 30 min. When compared to the control, the supernatant culture media of UCs exposed to hydrostatic pressure (10-15 cmH2O) exhibited a significant increase in ATP. In the absence of extracellular calcium, ATP release due to hydrostatic pressure was attenuated. Pharmacologically blocking transient receptor potential (TRP) channels, stretch-activated channels (SACs), and the epithelial sodium channel (ENaC) all abolished the hydrostatic pressure-evoked ATP release. These results provided evidence for the first time that cultured UCs are sensitive to physiologically relevant levels of hydrostatic pressure and that one or multiple mechanosensitive ion channels play a role in the mechanotransduction of hydrostatic pressure. These findings support the view that not only tissue stretch or tension, but also pressure is an important parameter for the sensing of bladder fullness. Second, UCs loaded with a fluorescence dye, calcein-AM were exposed to hydrostatic pressure up to 20 cmH2O while fluorescence intensity was measured in real-time. UCs exposed to hydrostatic pressure exhibited a sharp decrease in fluorescence intensity, indicative of a cell volume increase. These observations were confirmed by confocal imaging of primary UCs, which displayed a 7.7% volume increase when exposed to 20 cmH2O. Exposing the cells to Na-free solution and blocking of ENaC during pressure application resulted in a significantly lower change in cell volume. These results provided part of a novel mechanism involving cell swelling for mechanotransduction of hydrostatic pressure and an explanation for the previously reported ENaC-dependent, pressure-evoked ATP release by UCs. Finally, UCs were exposed to hydrostatic pressure while monitoring the kinetics of ATP release. UCs exposed to pressure (10 cmH2O) exhibited a sharp increase in ATP release that slowly decreased over time, while remaining elevated compared to baseline levels. This response was inhibited by blocking TRPV4 and ENaC. When UCs were exposed to a hypotonic stimulus, a sharp increase in ATP was exhibited followed by an immediate decrease to baseline levels. This increase in ATP was inhibited when blocking TRPV4, but was still present, albeit attenuated compared to the unblocked cells, when blocking ENaC. These results suggest that both ENaC and TRPV4 activation are necessary for ATP release during exposure to hydrostatic pressure. In addition, the osmotic shock results suggest that ENaC is upstream of the cell swelling response, while TRPV4 activation occurs between cell swelling and the ATP release. In summary, the results of the present research provide evidence that UCs respond to hydrostatic pressure with an increase in cell volume, activation of multiple mechanosensitive ion channels, and elevated ATP release. We have identified a possible mechanism by which UCs detect pressure and are the first to show that UCs respond to hydrostatic pressure in the absence of tissue stretch, which provides a novel contribution to cell mechanobiology that hydrostatic pressure alone is an important parameter involved in bladder mechanotransduction. This knowledge, in turn, will help elucidate the mechanism by which UCs, in normal and pathological cases, sense bladder fullness and ultimately aide in treatment of bladder disorders such as OAB

Mitochondria in endothelial cells: Sensors and integrators of environmental cues

The involvement of angiogenesis in disease and its potential as a therapeutic target have been firmly established over recent decades. Endothelial cells (ECs) are central elements in vessel homeostasis and regulate the passage of material and cells into and out of the bloodstream. EC proliferation and migration are modified by alterations to mitochondrial biogenesis and dynamics resulting from several signals and environmental cues, such as oxygen, hemodynamics, and nutrients. As intermediary signals, mitochondrial ROS are released as important downstream modulators of the expression of angiogenesis-related genes. In this review, we discuss the physiological actions of these signals and aberrant responses during vascular disorders.We thank M. M. Munoz-Hernandez and Dr Concepcion Jimenez for technical assistance, Keren Lopez Fernandez for the artwork in Fig. 1 and Simon Bartlett for English editing. This study was supported by MINECO: SAF2015-65633-R MSCA-COFUND-DP Doctoral programmes 2014. The CNIC is supported by MINECO and Pro-CNIC Foundation, and is a SO-MINECO (award SEV-2015-0505).S