Hyperspectral imaging and dynamic region of interest tracking approaches to quantify localized cAMP signals.

Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger known to orchestrate a myriad of cellular functions over a wide range of timescales. In the last 20 years, a variety of single-cell sensors have been developed to measure second messenger signals including cAMP, Ca2+, and the balance of kinase and phosphatase activities. These sensors utilize changes in fluorescence emission of an individual fluorophore or Förster resonance energy transfer (FRET) to detect changes in second messenger concentration. cAMP and kinase activity reporter probes have provided powerful tools for the study of localized signals. Studies relying on these and related probes have the potential to further revolutionize our understanding of G protein-coupled receptor signaling systems. Unfortunately, investigators have not been able to take full advantage of the potential of these probes due to the limited signal-to-noise ratio of the probes and the limited ability of standard epifluorescence and confocal microscope systems to simultaneously measure the distributions of multiple signals (e.g. cAMP, Ca2+, and changes in kinase activities) in real time. In this review, we focus on recently implemented strategies to overcome these limitations: hyperspectral imaging and adaptive thresholding approaches to track dynamic regions of interest (ROI). This combination of approaches increases signal-to-noise ratio and contrast, and allows identification of localized signals throughout cells. These in turn lead to the identification and quantification of intracellular signals with higher effective resolution. Hyperspectral imaging and dynamic ROI tracking approaches offer investigators additional tools with which to visualize and quantify multiplexed intracellular signaling systems.

Phosphodiesterase 4 mediates interleukin-8-induced heterologous desensitization of the ß2 -adrenergic receptor.

Acute respiratory distress syndrome (ARDS) is a life-threatening illness characterized by decreased alveolar-capillary barrier function, pulmonary edema consisting of proteinaceous fluid, and inhibition of net alveolar fluid transport responsible for resolution of pulmonary edema. There is currently no pharmacotherapy that has proven useful to prevent or treat ARDS, and two trials using beta-agonist therapy to treat ARDS demonstrated no effect. Prior studies indicated that IL-8-induced heterologous desensitization of the beta2-adrenergic receptor (ß2 -AR) led to decreased beta-agonist-induced mobilization of cyclic adenosine monophosphate (cAMP). Interestingly, phosphodiesterase (PDE) 4 inhibitors have been used in human airway diseases characterized by low intracellular cAMP levels and increases in specific cAMP hydrolyzing activity. Therefore, we hypothesized that PDE4 would mediate IL-8-induced heterologous internalization of the ß2 -AR and that PDE4 inhibition would restore beta-agonist-induced functions. We determined that CINC-1 (a functional IL-8 analog in rats) induces internalization of ß2 -AR from the cell surface, and arrestin-2, PDE4, and ß2 -AR form a complex during this process. Furthermore, we determined that cAMP associated with the plasma membrane was adversely affected by ß2 -AR heterologous desensitization. Additionally, we determined that rolipram, a PDE4 inhibitor, reversed CINC-1-induced derangements of cAMP and also caused ß2 -AR to successfully recycle back to the cell surface. Finally, we demonstrated that rolipram could reverse CINC-1-mediated inhibition of beta-agonist-induced alveolar fluid clearance in a murine model of trauma-shock. These results indicate that PDE4 plays a role in CINC-1-induced heterologous internalization of the ß2 -AR; PDE4 inhibition reverses these effects and may be a useful adjunct in particular ARDS patients.

cAMP signaling primes lung endothelial cells to activate caspase-1 during Pseudomonas aeruginosa infection.

Activation of the inflammasome-caspase-1 axis in lung endothelial cells is emerging as a novel arm of the innate immune response to pneumonia and sepsis caused by Pseudomonas aeruginosa. Increased levels of circulating autacoids are hallmarks of pneumonia and sepsis and induce physiological responses via cAMP signaling in targeted cells. However, it is unknown whether cAMP affects other functions, such as P. aeruginosa-induced caspase-1 activation. Herein, we describe the effects of cAMP signaling on caspase-1 activation using a single cell flow cytometry-based assay. P. aeruginosa infection of cultured lung endothelial cells caused caspase-1 activation in a distinct population of cells. Unexpectedly, pharmacological cAMP elevation increased the total number of lung endothelial cells with activated caspase-1. Interestingly, addition of cAMP agonists augmented P. aeruginosa infection of lung endothelial cells as a partial explanation underlying cAMP priming of caspase-1 activation. The cAMP effect(s) appeared to function as a priming signal because addition of cAMP agonists was required either before or early during the onset of infection. However, absolute cAMP levels measured by ELISA were not predictive of cAMP-priming effects. Importantly, inhibition of de novo cAMP synthesis decreased the number of lung endothelial cells with activated caspase-1 during infection. Collectively, our data suggest that lung endothelial cells rely on cAMP signaling to prime caspase-1 activation during P. aeruginosa infection.

Development of an endothelial cell-restricted transgenic reporter rat: a resource for physiological studies of vascular biology.

Here, we report the generation of a Cre-recombinase (iCre) transgenic rat, where iCre is driven using a vascular endothelial-cadherin (CDH5) promoter. The CDH5 promoter was cloned from rat pulmonary microvascular endothelial cells and demonstrated ~60% similarity to the murine counterpart. The cloned rat promoter was 2,508 bp, it extended 79 bp beyond the transcription start site, and it was 22,923 bp upstream of the translation start site. The novel promoter was cloned upstream of codon-optimized iCre and subcloned into a Sleeping Beauty transposon vector for transpositional transgenesis in Sprague-Dawley rats. Transgenic founders were generated and selected for iCre expression. Crossing the CDH5-iCre rat with a tdTomato reporter rat resulted in progeny displaying endothelium-restricted fluorescence. tdTomato fluorescence was prominent in major arteries and veins, and it was similar in males and females. Quantitative analysis of the carotid artery and the jugular vein revealed that, on average, more than 50% of the vascular surface area exhibited strong fluorescence. tdTomato fluorescence was observed in the circulations of every tissue tested. The microcirculation in all tissues tested displayed homogenous fluorescence. Fluorescence was examined across young (6-7.5 mo), middle (14-16.5 mo), and old age (17-19.5 mo) groups. Although tdTomato fluorescence was seen in middle- and old-age animals, the intensity of the fluorescence was significantly reduced compared with that seen in the young rats. Thus, this endothelium-restricted transgenic rat offers a novel platform to test endothelial microheterogeneity within all vascular segments, and it provides exceptional resolution of endothelium within-organ microcirculation for application to translational disease models.NEW & NOTEWORTHY The use of transgenic mice has been instrumental in advancing molecular insight of physiological processes, yet these models oftentimes do not faithfully recapitulate human physiology and pathophysiology. Rat models better replicate some human conditions, like Group 1 pulmonary arterial hypertension. Here, we report the development of an endothelial cell-restricted transgenic reporter rat that has broad application to vascular biology. This first-in-kind model offers exceptional endothelium-restricted tdTomato expression, in both conduit vessels and the microcirculations of organs.

Human ASIC1a mediates stronger acid-induced responses as compared with mouse ASIC1a.

Acid-sensing ion channels (ASICs) are the major proton receptor in the brain and a key mediator of acidosis-induced neuronal injuries in disease. Most of published data on ASIC function came from studies performed in mice, and relatively little is known about potential differences between human and mouse ASICs (hASIC and mASIC, respectively). This information is critical for us to better interpret the functional importance of ASICs in human disease. Here, we examined the expression of ASICs in acutely resected human cortical tissue. Compared with mouse cortex, human cortical tissue showed a similar ratio of ASIC1a:ASIC2a expression, had reduced ASIC2b level, and exhibited a higher membrane:total ratio of ASIC1a. We further investigated the mechanism for higher surface trafficking of hASIC1a in heterologous cells. A single amino acid at position 285 was critical for increased N-glycosylation and surface expression of hASIC1a. Consistent with the changes in trafficking and current, cells expressing hASIC1a or mASIC1a S285P mutant had a higher acid-activated calcium increase and exhibited worsened acidotoxicity. These data suggest that ASICs are likely to have a larger impact on acidosis-induced neuronal injuries in humans than mice, and this effect is, at least in part, a result of more efficient trafficking of hASIC1a.-Xu, Y., Jiang, Y.-Q., Li, C., He, M., Rusyniak, W. G., Annamdevula, N., Ochoa, J., Leavesley, S. J., Xu, J., Rich, T. C., Lin, M. T., Zha, X.-M. Human ASIC1a mediates stronger acid-induced responses as compared with mouse ASIC1a.

Spectral imaging of FRET-based sensors reveals sustained cAMP gradients in three spatial dimensions.

Cyclic AMP is a ubiquitous second messenger that orchestrates a variety of cellular functions over different timescales. The mechanisms underlying specificity within this signaling pathway are still not well understood. Several lines of evidence suggest the existence of spatial cAMP gradients within cells, and that compartmentalization underlies specificity within the cAMP signaling pathway. However, to date, no studies have visualized cAMP gradients in three spatial dimensions (3D: x, y, z).This is in part due to the limitations of FRET-based cAMP sensors, specifically the low signal-to-noise ratio intrinsic to all intracellular FRET probes. Here, we overcome this limitation, at least in part, by implementing spectral imaging approaches to estimate FRET efficiency when multiple fluorescent labels are used and when signals are measured from weakly expressed fluorescent proteins in the presence of background autofluorescence and stray light. Analysis of spectral image stacks in two spatial dimensions (2D) from single confocal slices indicates little or no cAMP gradients formed within pulmonary microvascular endothelial cells (PMVECs) under baseline conditions or following 10 min treatment with the adenylyl cyclase activator forskolin. However, analysis of spectral image stacks in 3D demonstrates marked cAMP gradients from the apical to basolateral face of PMVECs. Results demonstrate that spectral imaging approaches can be used to assess cAMP gradients-and in general gradients in fluorescence and FRET-within intact cells. Results also demonstrate that 2D imaging studies of localized fluorescence signals and, in particular, cAMP signals, whether using epifluorescence or confocal microscopy, may lead to erroneous conclusions about the existence and/or magnitude of gradients in either FRET or the underlying cAMP signals. Thus, with the exception of cellular structures that can be considered in one spatial dimension, such as neuronal processes, 3D measurements are required to assess mechanisms underlying compartmentalization and specificity within intracellular signaling pathways.

Pseudomonas aeruginosa infection liberates transmissible, cytotoxic prion amyloids.

Patients who recover from pneumonia subsequently have elevated rates of death after hospital discharge as a result of secondary organ damage, the causes of which are unknown. We used the bacterium Pseudomonas aeruginosa, a common cause of hospital-acquired pneumonia, as a model for investigating this phenomenon. We show that infection of pulmonary endothelial cells by P. aeruginosa induces production and release of a cytotoxic amyloid molecule with prion characteristics, including resistance to various nucleases and proteases. This cytotoxin was self-propagating, was neutralized by anti-amyloid Abs, and induced death of endothelial cells and neurons. Moreover, the cytotoxin induced edema in isolated lungs. Endothelial cells and isolated lungs were protected from cytotoxin-induced death by stimulation of signal transduction pathways that are linked to prion protein. Analysis of bronchoalveolar lavage fluid collected from human patients with P. aeruginosa pneumonia demonstrated cytotoxic activity, and lavage fluid contained amyloid molecules, including oligomeric τ and Aß. Demonstration of long-lived cytotoxic agents after Pseudomonas infection may establish a molecular link to the high rates of death as a result of end-organ damage in the months after recovery from pneumonia, and modulation of signal transduction pathways that have been linked to prion protein may provide a mechanism for intervention.-Balczon, R., Morrow, K. A., Zhou, C., Edmonds, B., Alexeyev, M., Pittet, J.-F., Wagener, B. M., Moser, S. A., Leavesley, S., Zha, X., Frank, D. W., Stevens, T. Pseudomonas aeruginosa infection liberates transmissible, cytotoxic prion amyloids.

Estimating the magnitude of near-membrane PDE4 activity in living cells.

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 µM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments.

High-speed fluorescence excitation-scanning hyperspectral imaging microscopy using thin-film tunable filters.

Hyperspectral imaging (HSI) technologies have enabled a range of experimental techniques and studies in the fluorescence microscopy field. Unfortunately, a drawback of many HSI microscope platforms is increased acquisition time required to collect images across many spectral bands, as well as signal loss due to the need to filter or disperse emitted fluorescence into many discrete bands. We have previously demonstrated that an alternative approach of scanning the fluorescence excitation spectrum can greatly improve system efficiency by decreasing light losses associated with emission filtering. Our initial system was configured using an array of thin-film tunable filters (TFTFs, VersaChrome, Semrock) mounted in a tiltable filter wheel (VF-5, Sutter) that required ~150-200 ms to switch between wavelengths. Here, we present a new configuration for high-speed switching of TFTFs to allow rapid time-lapse HSI microscopy. A TFTF array was mounted in a custom holder that was attached to a piezoelectric rotation mount (ThorLabs), allowing high-speed rotation. Switching between adjacent filters was achieved using the internal optics of a DG-4 lightsource (Sutter Instrument), including a pair of off-axis parabolic mirrors and galvanometers. Output light was coupled to a liquid lightguide and into an inverted widefield fluorescence microscope (TI-2, Nikon Instruments). Initial tests indicate that the HSI system provides a 15-20 nm bandwidth tunable excitation band and ~10-20 ms wavelength switch time, allowing for high-speed HSI imaging of dynamic cellular events. This work was supported by NIH P01HL066299, R01HL169522, NIH TL1TR003106, and NSF MRI1725937.

Fluorescence excitation-scanning hyperspectral imaging with scalable 2D-3D deep learning framework for colorectal cancer detection.

Colorectal cancer is one of the top contributors to cancer-related deaths in the United States, with over 100,000 estimated cases in 2020 and over 50,000 deaths. The most common screening technique is minimally invasive colonoscopy using either reflected white light endoscopy or narrow-band imaging. However, current imaging modalities have only moderate sensitivity and specificity for lesion detection. We have developed a novel fluorescence excitation-scanning hyperspectral imaging (HSI) approach to sample image and spectroscopic data simultaneously on microscope and endoscope platforms for enhanced diagnostic potential. Unfortunately, fluorescence excitation-scanning HSI datasets pose major challenges for data processing, interpretability, and classification due to their high dimensionality. Here, we present an end-to-end scalable Artificial Intelligence (AI) framework built for classification of excitation-scanning HSI microscopy data that provides accurate image classification and interpretability of the AI decision-making process. The developed AI framework is able to perform real-time HSI classification with different speed/classification performance trade-offs by tailoring the dimensionality of the dataset, supporting different dimensions of deep learning models, and varying the architecture of deep learning models. We have also incorporated tools to visualize the exact location of the lesion detected by the AI decision-making process and to provide heatmap-based pixel-by-pixel interpretability. In addition, our deep learning framework provides wavelength-dependent impact as a heatmap, which allows visualization of the contributions of HSI wavelength bands during the AI decision-making process. This framework is well-suited for HSI microscope and endoscope platforms, where real-time analysis and visualization of classification results are required by clinicians.

Assessing FRET using spectral techniques.

Förster resonance energy transfer (FRET) techniques have proven invaluable for probing the complex nature of protein-protein interactions, protein folding, and intracellular signaling events. These techniques have traditionally been implemented with the use of one or more fluorescence band-pass filters, either as fluorescence microscopy filter cubes, or as dichroic mirrors and band-pass filters in flow cytometry. In addition, new approaches for measuring FRET, such as fluorescence lifetime and acceptor photobleaching, have been developed. Hyperspectral techniques for imaging and flow cytometry have also shown to be promising for performing FRET measurements. In this study, we have compared traditional (filter-based) FRET approaches to three spectral-based approaches: the ratio of acceptor-to-donor peak emission, linear spectral unmixing, and linear spectral unmixing with a correction for direct acceptor excitation. All methods are estimates of FRET efficiency, except for one-filter set and three-filter set FRET indices, which are included for consistency with prior literature. In the first part of this study, spectrofluorimetric data were collected from a CFP-Epac-YFP FRET probe that has been used for intracellular cAMP measurements. All comparisons were performed using the same spectrofluorimetric datasets as input data, to provide a relevant comparison. Linear spectral unmixing resulted in measurements with the lowest coefficient of variation (0.10) as well as accurate fits using the Hill equation. FRET efficiency methods produced coefficients of variation of less than 0.20, while FRET indices produced coefficients of variation greater than 8.00. These results demonstrate that spectral FRET measurements provide improved response over standard, filter-based measurements. Using spectral approaches, single-cell measurements were conducted through hyperspectral confocal microscopy, linear unmixing, and cell segmentation with quantitative image analysis. Results from these studies confirmed that spectral imaging is effective for measuring subcellular, time-dependent FRET dynamics and that additional fluorescent signals can be readily separated from FRET signals, enabling multilabel studies of molecular interactions. © 2013 International Society for Advancement of Cytometry.

An approach for characterizing and comparing hyperspectral microscopy systems.

Hyperspectral imaging and analysis approaches offer accurate detection and quantification of fluorescently-labeled proteins and cells in highly autofluorescent tissues. However, selecting optimum acquisition settings for hyperspectral imaging is often a daunting task. In this study, we compared two hyperspectral systems-a widefield system with acoustic optical tunable filter (AOTF) and charge coupled device (CCD) camera, and a confocal system with diffraction gratings and photomultiplier tube (PMT) array. We measured the effects of system parameters on hyperspectral image quality and linear unmixing results. Parameters that were assessed for the confocal system included pinhole diameter, laser power, PMT gain and for the widefield system included arc lamp intensity, and camera gain. The signal-to-noise ratio (SNR) and the root-mean-square error (RMS error) were measured to assess system performance. Photobleaching dynamics were studied. Finally, theoretical sensitivity studies were performed to estimate the incremental response (sensitivity) and false-positive detection rates (specificity). Results indicate that hyperspectral imaging assays are highly dependent on system parameters and experimental conditions. For detection of green fluorescent protein (GFP)-expressing cells in fixed lung tissues, a confocal pinhole of five airy disk units, high excitation intensity and low detector gain were optimal. The theoretical sensitivity studies revealed that widefield hyperspectral microscopy was able to detect GFP with fewer false positive occurrences than confocal microscopy, even though confocal microscopy offered improved signal and noise characteristics. These studies provide a framework for optimization that can be applied to a variety of hyperspectral imaging systems.

Multifaceted mirror array illuminator for fluorescence excitation-scanning spectral imaging microscopy.

Significance: Hyperspectral imaging (HSI) technologies offer great potential in fluorescence microscopy for multiplexed imaging, autofluorescence removal, and analysis of autofluorescent molecules. However, there are also associated trade-offs when implementing HSI in fluorescence microscopy systems, such as decreased acquisition speed, resolution, or field-of-view due to the need to acquire spectral information in addition to spatial information. The vast majority of HSI fluorescence microscopy systems provide spectral discrimination by filtering or dispersing the fluorescence emission, which may result in loss of emitted fluorescence signal due to optical filters, dispersive optics, or supporting optics, such as slits and collimators. Technologies that scan the fluorescence excitation spectrum may offer an approach to mitigate some of these trade-offs by decreasing the complexity of the emission light path. Aim: We describe the development of an optical technique for hyperspectral imaging fluorescence excitation-scanning (HIFEX) on a microscope system. Approach: The approach is based on the design of an array of wavelength-dependent light emitting diodes (LEDs) and a unique beam combining system that uses a multifurcated mirror. The system was modeled and optimized using optical ray trace simulations, and a prototype was built and coupled to an inverted microscope platform. The prototype system was calibrated, and initial feasibility testing was performed by imaging multilabel slide preparations. Results: We present results from optical ray trace simulations, prototyping, calibration, and feasibility testing of the system. Results indicate that the system can discriminate between at least six fluorescent labels and autofluorescence and that the approach can provide decreased wavelength switching times, in comparison with mechanically tuned filters. Conclusions: We anticipate that LED-based HIFEX microscopy may provide improved performance for time-dependent and photosensitive assays.

Comparing Performance of Spectral Image Analysis Approaches for Detection of Cellular Signals in Time-Lapse Hyperspectral Imaging Fluorescence Excitation-Scanning Microscopy.

Hyperspectral imaging (HSI) technology has been applied in a range of fields for target detection and mixture analysis. While HSI was originally developed for remote sensing applications, modern uses include agriculture, historical document authentication, and medicine. HSI has also shown great utility in fluorescence microscopy. However, traditional fluorescence microscopy HSI systems have suffered from limited signal strength due to the need to filter or disperse the emitted light across many spectral bands. We have previously demonstrated that sampling the fluorescence excitation spectrum may provide an alternative approach with improved signal strength. Here, we report on the use of excitation-scanning HSI for dynamic cell signaling studies-in this case, the study of the second messenger Ca2+. Time-lapse excitation-scanning HSI data of Ca2+ signals in human airway smooth muscle cells (HASMCs) were acquired and analyzed using four spectral analysis algorithms: linear unmixing (LU), spectral angle mapper (SAM), constrained energy minimization (CEM), and matched filter (MF), and the performances were compared. Results indicate that LU and MF provided similar linear responses to increasing Ca2+ and could both be effectively used for excitation-scanning HSI. A theoretical sensitivity framework was used to enable the filtering of analyzed images to reject pixels with signals below a minimum detectable limit. The results indicated that subtle kinetic features might be revealed through pixel filtering. Overall, the results suggest that excitation-scanning HSI can be employed for kinetic measurements of cell signals or other dynamic cellular events and that the selection of an appropriate analysis algorithm and pixel filtering may aid in the extraction of quantitative signal traces. These approaches may be especially helpful for cases where the signal of interest is masked by strong cellular autofluorescence or other competing signals.

Combined hyperspectral imaging, monolayer stress microscopy, and S8 image analysis approaches for simultaneously interrogating cellular signals and biomechanics.

Second messenger signals, e.g., Ca2+ and cyclic nucleotides, orchestrate a wide range of cellular events. The methods by which second messenger signals determine specific physiological responses are complex. Recent studies point to the importance of temporal and spatial encoding in determining signal specificity. Studies also indicate the importance of mechanical stimuli, substrate stiffness, and mechanical responses - the "mechanosome" - in regulating physiology. Hence, approaches that probe both chemical and mechanical signals are needed. Here, we report preliminary efforts to combine hyperspectral imaging for second messenger signal measurements, monolayer stress microscopy for mechanical force measurements, and S8 analysis software for quantifying localized signals - specifically, Ca2+ dynamics and mechanical forces in human airway smooth muscle cells (HASMCs). HASMCs were prepared as confluent monolayers on 11 kPa gels with embedded fluorescent microparticles that serve as fiducial markers as well as smaller microparticles to measure deformation (strain). Imaging was performed using a custom excitation-scanning hyperspectral microscope. Hyperspectral images were unmixed to identify signals from cellular fluorescent labels (e.g., CAL 590-AM) and fluorescent microparticles. Images were analyzed to quantify localized force dynamics through monolayer stress microscopy. S8 software was used to identify, track, and quantify spatially-localized Ca2+ activity. Results indicate that localized and transient cellular signals and forces can be quantified and mapped within cell populations. Importantly, these results establish a method for simultaneous interrogation of cellular signals and mechanical forces that may play synergistic roles in regulating downstream cellular physiology in confluent monolayers. This work was supported by NIH P01HL066299, R01HL137030, R01HL058506, and NSF MRI1725937. Drs. Leavesley and Rich disclose financial interest in a university start-up company, SpectraCyte LLC, to commercialize spectral imaging technologies.

Assessment of cellular mechanisms contributing to cAMP compartmentalization in pulmonary microvascular endothelial cells.

Cyclic AMP signals encode information required to differentially regulate a wide variety of cellular responses; yet it is not well understood how information is encrypted within these signals. An emerging concept is that compartmentalization underlies specificity within the cAMP signaling pathway. This concept is based on a series of observations indicating that cAMP levels are distinct in different regions of the cell. One such observation is that cAMP production at the plasma membrane increases pulmonary microvascular endothelial barrier integrity, whereas cAMP production in the cytosol disrupts barrier integrity. To better understand how cAMP signals might be compartmentalized, we have developed mathematical models in which cellular geometry as well as total adenylyl cyclase and phosphodiesterase activities were constrained to approximate values measured in pulmonary microvascular endothelial cells. These simulations suggest that the subcellular localizations of adenylyl cyclase and phosphodiesterase activities are by themselves insufficient to generate physiologically relevant cAMP gradients. Thus, the assembly of adenylyl cyclase, phosphodiesterase, and protein kinase A onto protein scaffolds is by itself unlikely to ensure signal specificity. Rather, our simulations suggest that reductions in the effective cAMP diffusion coefficient may facilitate the formation of substantial cAMP gradients. We conclude that reductions in the effective rate of cAMP diffusion due to buffers, structural impediments, and local changes in viscosity greatly facilitate the ability of signaling complexes to impart specificity within the cAMP signaling pathway.

Endoscopy Lifetime Systems Architecture: Scoping Out the Past to Diagnose the Future Technology.

Systems engineering captures the desires and needs of the customer to conceptualize a system from the overall goal down to the small details prior to any physical development. While many systems projects tend to be large and complicated (i.e., cloud-based infrastructure, long-term space travel shuttles, missile defense systems), systems engineering can also be applied to smaller, complex systems. Here, the system of interest is the endoscope, a standard biomedical screening device used in laparoscopic surgery, screening of upper and lower gastrointestinal tracts, and inspection of the upper airway. Often, endoscopic inspection is used to identify pre-cancerous and cancerous tissues, and hence, a requirement for endoscopic systems is the ability to provide images with high contrast between areas of normal tissue and neoplasia (early-stage abnormal tissue growth). For this manuscript, the endoscope was reviewed for all the technological advancements thus far to theorize what the next version of the system could be in order to provide improved detection capabilities. Endoscopic technology was decomposed into categories, using systems architecture and systems thinking, to visualize the improvements throughout the system's lifetime from the original to current state-of-the-art. Results from this review were used to identify trends in subsystems and components to estimate the theoretical performance maxima for different subsystems as well as areas for further development. The subsystem analysis indicated that future endoscope systems will focus on more complex imaging and higher computational requirements that will provide improved contrast in order to have higher accuracy in optical diagnoses of early, abnormal tissue growth.