Project description:Previously unobtainable measurements of alveolar pH were obtained using an endoscope-deployable optrode. The pH sensing was achieved using functionalized gold nanoshell sensors and surface enhanced Raman spectroscopy (SERS). The optrode consisted of an asymmetric dual-core optical fiber designed for spatially separating the optical pump delivery and signal collection, in order to circumvent the unwanted Raman signal generated within the fiber. Using this approach, we demonstrate a ~100-fold increase in SERS signal-to-fiber background ratio, and demonstrate multiple site pH sensing with a measurement accuracy of ± 0.07 pH units in the respiratory acini of an ex vivo ovine lung model. We also demonstrate that alveolar pH changes in response to ventilation.
Project description:This series compares the effects that two pharmacologically distinct ligands at the mitochondrial peripheral-type benzodiazepine receptor (Bzrp) has on gene expression in early mouse embryos cultured under different oxygen levels. Mouse embryos averaging 16-18 somite pairs were explanted on 9 d.p.c. and grown in culture under optimal oxygenation (21% oxygen, hyperbaric with respect to arterial blood = 80-100 mmHg). Most embryos (>90%) develop normally in over a 24h culture period. When embryos receive suboptimal oxygenation for 2.0h (5% oxygen, similar to venous blood = 38-40 mmHg) a high percentage of them (>60%) go on to develop abnormally or else fail. These hypoxic embryos are remediated with 5 uM PK11195 to ~25% malformation rate. Cultured embryos were exposed to different oxygen levels (5% or 21%) in the presence of the Bzrp ligands PK11195 (Bzrp antagonist) versus Ro5-4864 (Bzrp agonist). The sampling time was guided by p53 protein induction with hypoxia, and remediation with PK11195, at 4.0h of culture and exposures. Thus all measurements were on RNA from the prosencephalon collected 2.0h after start of the test period on 9 d.p.c. Keywords = peripheral benzodiazepine receptor Keywords = oxygen sensing Keywords = Bzrp ligands Keywords = PK11195 Keywords = Ro5-4864 Keywords = embryo Keywords = p53 protein induction Keywords: ordered
Project description:Bronchopulmonary dysplasia and emphysema are life-threatening diseases resulting from impaired alveolar development or alveolar destruction. Both conditions lack effective therapies. Angiogenic growth factors promote alveolar growth and contribute to alveolar maintenance. Endothelial colony-forming cells (ECFCs) represent a subset of circulating and resident endothelial cells capable of self-renewal and de novo vessel formation. We hypothesized that resident ECFCs exist in the developing lung, that they are impaired during arrested alveolar growth in experimental bronchopulmonary dysplasia, and that exogenous ECFCs restore disrupted alveolar growth.Human fetal and neonatal rat lungs contain ECFCs with robust proliferative potential, secondary colony formation on replating, and de novo blood vessel formation in vivo when transplanted into immunodeficient mice. In contrast, human fetal lung ECFCs exposed to hyperoxia in vitro and neonatal rat ECFCs isolated from hyperoxic alveolar growth-arrested rat lungs mimicking bronchopulmonary dysplasia proliferated less, showed decreased clonogenic capacity, and formed fewer capillary-like networks. Intrajugular administration of human cord blood-derived ECFCs after established arrested alveolar growth restored lung function, alveolar and lung vascular growth, and attenuated pulmonary hypertension. Lung ECFC colony- and capillary-like network-forming capabilities were also restored. Low ECFC engraftment and the protective effect of cell-free ECFC-derived conditioned media suggest a paracrine effect. Long-term (10 months) assessment of ECFC therapy showed no adverse effects with persistent improvement in lung structure, exercise capacity, and pulmonary hypertension.Impaired ECFC function may contribute to arrested alveolar growth. Cord blood-derived ECFC therapy may offer new therapeutic options for lung diseases characterized by alveolar damage.
Project description:This series compares the effects that two pharmacologically distinct ligands at the mitochondrial peripheral-type benzodiazepine receptor (Bzrp) has on gene expression in early mouse embryos cultured under different oxygen levels. Mouse embryos averaging 16-18 somite pairs were explanted on 9 d.p.c. and grown in culture under optimal oxygenation (21% oxygen, hyperbaric with respect to arterial blood = 80-100 mmHg). Most embryos (>90%) develop normally in over a 24h culture period. When embryos receive suboptimal oxygenation for 2.0h (5% oxygen, similar to venous blood = 38-40 mmHg) a high percentage of them (>60%) go on to develop abnormally or else fail. These hypoxic embryos are remediated with 5 uM PK11195 to ~25% malformation rate. Cultured embryos were exposed to different oxygen levels (5% or 21%) in the presence of the Bzrp ligands PK11195 (Bzrp antagonist) versus Ro5-4864 (Bzrp agonist). The sampling time was guided by p53 protein induction with hypoxia, and remediation with PK11195, at 4.0h of culture and exposures. Thus all measurements were on RNA from the prosencephalon collected 2.0h after start of the test period on 9 d.p.c. Keywords = peripheral benzodiazepine receptor Keywords = oxygen sensing Keywords = Bzrp ligands Keywords = PK11195 Keywords = Ro5-4864 Keywords = embryo Keywords = p53 protein induction
Project description:Molecular oxygen has profound effects on cell and tissue viability. Relevant sensor forms that can rapidly determine dissolved oxygen levels under biologically relevant conditions provide critical metabolic information. Using 0.5 ?m diameter electrospun polycaprolactone (PCL) fiber containing an oxygen-sensitive probe, tris (4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride, we observed a response time of 0.9±0.12 s while the t95 for the corresponding film was more than two orders of magnitude greater. Interestingly, the response and recovery times of slightly larger diameter PCL fibers were 1.79±0.23 s and 2.29±0.13 s, respectively, while the recovery time was not statistically different likely due to the more limited interactions of nitrogen with the polymer matrix. A more than 10-fold increase in PCL fiber diameter reduces oxygen sensitivity while having minor effects on response time; conversely, decreases in fiber diameter to less than 0.5 ?m would likely decrease response times even further. In addition, a 50°C heat treatment of the electrospun fiber resulted in both increased Stern-Volmer slope and linearity likely due to secondary recrystallization that further homogenized the probe microenvironment. At exposure times up to 3600 s in length, photobleaching was observed but was largely eliminated by the use of either polyethersulfone (PES) or a PES-PCL core-shell composition. However, this resulted in 2- and 3-fold slower response times. Finally, even the non-core shell compositions containing the Ru oxygen probe result in no apparent cytotoxicity in representative glioblastoma cell populations.
Project description:Dual emissive luminescence properties of solid-state difluoroboron β-diketonate-poly(lactic acid) (BF2bdk-PLA) materials have been utilized as biological oxygen sensors. Dyes with red-shifted absorption and emission are important for multiplexing and in vivo imaging, thus hydroxyl-functionalized dinaphthoylmethane initiators and dye-PLA conjugates BF2dnm(X)PLA (X = H, Br, I) with extended conjugation were synthesized. The luminescent materials show red-shifted absorbance (~435 nm) and fluorescence tunability by molecular weight. Fluorescence colors range from yellow (~530 nm) in 10 - 12 kDa polymers to green (~490 nm) in 20 - 30 kDa polymers. Room-temperature phosphorescence (RTP) and thermally activated delayed fluorescence (TADF) are present under a nitrogen atmosphere. For the iodine-substituted derivative, BF2dnm(I)PLA, clearly distinguishable fluorescence (green) and phosphorescence (orange) peaks are present, making it ideal for ratiometric oxygen-sensing and imaging. Bromide and hydrogen analogues with weaker relative phosphorescence intensities and longer phosphorescence lifetimes can be used as highly sensitive, concentration independent, lifetime-based oxygen sensors or for gated emission detection. BF2dnm(I)PLA nanoparticles were taken up by T41 mouse mammary cells and successfully demonstrated differences in vitro ratiometric measurement of oxygen.
Project description:Arterial oxygen partial pressure can increase during inspiration and decrease during expiration in the presence of a variable shunt fraction, such as with cyclical atelectasis, but it is generally presumed to remain constant within a respiratory cycle in the healthy lung. We measured arterial oxygen partial pressure continuously with a fast intra-vascular sensor in the carotid artery of anaesthetized, mechanically ventilated pigs, without lung injury. Here we demonstrate that arterial oxygen partial pressure shows respiratory oscillations in the uninjured pig lung, in the absence of cyclical atelectasis (as determined with dynamic computed tomography), with oscillation amplitudes that exceeded 50?mmHg, depending on the conditions of mechanical ventilation. These arterial oxygen partial pressure respiratory oscillations can be modelled from a single alveolar compartment and a constant oxygen uptake, without the requirement for an increased shunt fraction during expiration. Our results are likely to contribute to the interpretation of arterial oxygen respiratory oscillations observed during mechanical ventilation in the acute respiratory distress syndrome.
Project description:ObjectiveAtherosclerosis-prone regions of arteries are characterized by complex flow patterns where the magnitude of shear stress is low and direction rapidly changes, termed disturbed flow. How endothelial cells sense flow direction and how it impacts inflammatory effects of disturbed flow are unknown. We therefore aimed to understand how endothelial cells respond to changes in flow direction.Approach and resultsUsing a recently developed flow system capable of changing flow direction to any angle, we show that responses of aligned endothelial cells are determined by flow direction relative to their morphological and cytoskeletal axis. Activation of the atheroprotective endothelial nitric oxide synthase pathway is maximal at 180° and undetectable at 90°, whereas activation of proinflammatory nuclear factor-κB is maximal at 90° and undetectable at 180°. Similar effects were observed in randomly oriented cells in naive monolayers subjected to onset of shear. Cells aligned on micropatterned substrates subjected to oscillatory flow were also examined. In this system, parallel flow preferentially activated endothelial nitric oxide synthase and production of nitric oxide, whereas perpendicular flow preferentially activated reactive oxygen production and nuclear factor-κB.ConclusionsThese data show that the angle between flow and the cell axis defined by their shape and cytoskeleton determines endothelial cell responses. The data also strongly suggest that the inability of cells to align in low and oscillatory flow is a key determinant of the resultant inflammatory activation.
Project description:The ability to sense and adapt to changes in pO2 is crucial for basic metabolism in most organisms, leading to elaborate pathways for sensing hypoxia (low pO2). This review focuses on the mechanisms utilized by mammals and bacteria to sense hypoxia. While responses to acute hypoxia in mammalian tissues lead to altered vascular tension, the molecular mechanism of signal transduction is not well understood. In contrast, chronic hypoxia evokes cellular responses that lead to transcriptional changes mediated by the hypoxia inducible factor (HIF), which is directly controlled by post-translational hydroxylation of HIF by the non-heme Fe(II)/αKG-dependent enzymes FIH and PHD2. Research on PHD2 and FIH is focused on developing inhibitors and understanding the links between HIF binding and the O2 reaction in these enzymes. Sulfur speciation is a putative mechanism for acute O2-sensing, with special focus on the role of H2S. This sulfur-centered model is discussed, as are some of the directions for further refinement of this model. In contrast to mammals, bacterial O2-sensing relies on protein cofactors that either bind O2 or oxidatively decompose. The sensing modality for bacterial O2-sensors is either via altered DNA binding affinity of the sensory protein, or else due to the actions of a two-component signaling cascade. Emerging data suggests that proteins containing a hemerythrin-domain, such as FBXL5, may serve to connect iron sensing to O2-sensing in both bacteria and humans. As specific molecular machinery becomes identified, these hypoxia sensing pathways present therapeutic targets for diseases including ischemia, cancer, or bacterial infection.
Project description:Mitochondria maintain a constant rate of aerobic respiration over a wide range of oxygen levels. However, the control strategies underlying oxygen homeostasis are still unclear. Using mathematical modeling, we found that the mitochondrial electron transport chain (ETC) responds to oxygen level changes by undergoing compensatory changes in reduced electron carrier levels. This emergent behavior, which we named cosubstrate compensation (CSC), enables the ETC to maintain homeostasis over a wide of oxygen levels. When performing CSC, our ETC models recapitulated a classic scaling relationship discovered by Chance [Chance B (1965) J. Gen. Physiol. 49:163-165] relating the extent of oxygen homeostasis to the kinetics of mitochondrial electron transport. Analysis of an in silico mitochondrial respiratory system further showed evidence that CSC constitutes the dominant control strategy for mitochondrial oxygen homeostasis during active respiration. Our findings indicate that CSC constitutes a robust control strategy for homeostasis and adaptation in cellular biochemical networks.