Do Pulmonary Cavity Shapes Influence Lung Function?

Distribution of lung tissue within the chest cavity is a key contributor to delivery of both blood and air to the gas exchange regions of the lung. This distribution is multifactorial with influences from parenchyma, gravity, and level of inflation. We hypothesize that the manner in which lung inflates, for example, the primarily diaphragmatic nature of normal breathing, is an important contributor to regional lung tissue distribution. To investigate this hypothesis, we present an organ-level model of lung tissue mechanics, which incorporates pleural cavity change due to change in lung volume or posture. We quantify the changes using shape and density metrics in ten healthy subjects scanned supine at end-inspiratory and end-expiratory volumes and ten subjects scanned at both supine and prone end-inspiratory volumes. Comparing end-expiratory to end-inspiratory volume, we see primarily a change in the cranial-caudal dimension of the lung, reflective of movement of diaphragm. In the diaphragmatic region, there is greater regional lung expansion than in the cranial aspect, which is restricted by the chest wall. When moving from supine to prone, a restriction of lung was observed anteriorly, resulting in a generally reduced lung volume and a redistribution of air volume posteriorly. In general, we see the highest in lung tissue density heterogeneity in regions of the lung that are most inflated. Using our computational model, we quantify the impact of pleural cavity shape change on regional lung distribution and predict the impact on regional elastic recoil pressure.

Impact of advanced detector technology and iterative reconstruction on low-dose quantitative assessment of lung computed tomography density in a biological lung model.

PURPOSE: Quantitative computed tomography (QCT)-derived measures of lung density are valued methods for objectively characterizing lung parenchymal and peripheral airways disease and are being used in a growing number of lung disease focused trials. Detector and reconstruction improvements in CT technology have allowed for significant radiation dose reduction in image acquisition with comparable qualitative image quality. We report the impact of detector type and reconstruction type on QCT lung density measures in relation to decreasing dose indices. METHODS: Two sets of studies were completed in an in vivo pig model with a SOMATOM Definition Flash CT system: (a) prior to system upgrade with conventional detectors (UFC) and filtered back projection (FBP), and (b) post system upgrade with integrated electronic detectors (STELLAR) and iterative reconstruction (SAFIRE). CT data were acquired across estimated CT volume dose indices (CTDIvol ) ranging from 0.75 to 15 mGy at both inspiratory and expiratory breath holds. Semiautomated lung segmentations allowed calculation of histogram median, kurtosis, and 15th percentile. Percentage of voxels below -910 HU and -950 HU (inspiratory), and -856 HU (expiratory) were also examined. The changes in these QCT metrics from dose reduction (15 mGy down to 0.75 mGy) were calculated relative to paired reference values (15 mGy). Results were compared based on detector and reconstruction type. RESULTS: In this study, STELLAR detectors improved concordance with 15 mGy values down to 3 mGy for inspiratory scans and 6 mGy for expiratory scans. The addition of SAFIRE reconstruction in all acquired measurements resulted in minimal deviation from reference values at 0.75 mGy. CONCLUSION: The use of STELLAR integrated electronic detectors and SAFIRE iterative reconstruction may allow for comparable lung density measures with CT dose indices down to 0.75 mGy.

Metrics of lung tissue heterogeneity depend on BMI but not age.

Altered parenchymal microstructure and complexity have been observed in older age. How to distinguish between healthy, expected changes and early signs of pathology remains poorly understood. An objective quantitative analysis of computed tomography imaging was conducted to compare mean lung density, tissue density distributions, and tissue heterogeneity in 16 subjects, 8 aged >60 yr who were gender and body mass index matched with 8 subjects aged <30 yr. Subjects had never been smokers, with no prior respiratory disease, and no radiologically identified abnormalities on computed tomography. Volume-controlled breath hold imaging acquired at 80% vital capacity (end inspiration) and 55% vital capacity (end expiration) were used for analysis. Mean lung density was not different between the age groups at end inspiration ( P = 0.806) but was larger in the younger group at end expiration (0.26 ± 0.033 vs. 0.22 ± 0.026, P = 0.008), as is expected due to increased air trapping in the older population. However, gravitational gradients of tissue density did not differ with age; the only difference in distribution of tissue density between the two age groups was a lower density in the apices of the older group at end expiration. The heterogeneity of the lung tissue assessed using two metrics showed significant differences between end inspiration and end expiration, no dependence on age, and a significant relationship with body mass index at both lung volumes when heterogeneity was calculated using quadtree decomposition but only at end expiration when using a fractal dimension. NEW & NOTEWORTHY Changes to lung tissue heterogeneity can be a normal part of aging but can also be an early indicator of disease. We use novel techniques, which have previously not been used on thoracic computed tomography imaging, to quantify lung tissue heterogeneity in young and old healthy subjects. Our results show no dependence on age but a significant correlation with body mass index.

Sinogram Affirmed Iterative Reconstruction (SAFIRE) versus weighted filtered back projection (WFBP) effects on quantitative measure in the COPDGene 2 test object.

PURPOSE: Assessing pulmonary emphysema using Quantitative CT of the lung depends on accurate measures of CT density. Sinogram-Affirmed-Iterative-Reconstruction (SAFIRE) is a new approach for reconstructing CT data acquired at significantly lower doses. However, quantitative effects of this method remain unexplored. The authors investigated the effects on the median values of materials in the COPDGene2 test-object as a function of the reconstruction method [weighted filtered back projection (WFBP) versus SAFIRE], test-object size, dose, and material composition using a Siemens SOMATOM Definition FLASH CT scanner. METHODS: The COPDGene2 test-object contains eight materials; acrylic, water, four foams (20 lb, 12 lb, lung-equivalent, and 4 lb emphysema-equivalent), internal and external-air. The test-object was scanned with three different outer ring sizes, simulating three different body habitus. There is an average size (36 cm) Ring A, large size (40 cm) Ring B, and small size Ring C (30 cm). The CT protocol used 120 kVp, 0.5 s rotation, 1.0 pitch, and a 0.6 slice collimation with progressively decreasing x-ray exposure values, 11.94-0.74 mGy. With a thorax length of 30 cm, the corresponding effective doses would be 5.01-0.31 mSv. The effects of using SAFIRE versus WFBP were assessed using a two tailed t-test for each ring size, material, and dose. Multivariable linear regression was used to evaluate the relative effects of ring size, material composition, dose, and reconstruction method on the measured median value in HU. RESULTS: SAFIRE versus WFBP, at the largest ring size and two lowest doses there was a significant difference in median values of 4 lb-foam, p<0.01. Using the smallest ring size at the lowest dose level there was a significant difference in the median value of 4 lb-foam, but the effect size was small, 1 HU. There is a significant difference in median values of both internal and external air using both the small and medium size rings at the three lowest dose levels, p<0.05. There are significant differences noted at both high and low dose levels when using the large ring size in the median values of internal and external air when, p<0.05. These effects on 4 lb-foam, inside and outside air are shown to be in part due to truncation effects on the median value since the lowest HU value in the CT scale used is -1024 HU. Multivariable linear regression results demonstrated significant effects on the measured material median value and standard deviation due to ring size, material composition, dose level, and reconstruction method, p<0.05. CONCLUSIONS: The authors have shown that there is no significant effect on the median values obtained when using WFBP versus SAFIRE in materials with CT density between 120 and -856 HU using three different test-object sizes and CT doses that vary from 11.94 to 0.74 mGy. The authors have demonstrated there are significant effects on median values obtained when using WFBP versus SAFIRE in materials with CT density values between -937 and -1000 HU depending on the ring size and dose used. As expected, there is considerable reduction in image noise (lower standard deviation) using SAFIRE versus WFBP with all ring sizes, doses, and materials in the COPDGene2 test-object.

Reference standard and statistical model for intersite and temporal comparisons of CT attenuation in a multicenter quantitative lung study.

PURPOSE: The purpose of this study was to detect and analyze anomalies between a large number of computed tomography (CT) scanners, tracked over time, utilized to collect human pulmonary CT data for a national multicenter study: chronic obstructive pulmonary disease genetic epidemiology study (COPDGene). METHODS: A custom designed CT reference standard "Test Object" has been developed to evaluate the relevant differences in CT attenuation between CT scanners in COPDGene. The materials used in the Test Object to assess CT scanner accuracy and precision included lung equivalent foam (-856 HU), internal air (-1000 HU), water (0 HU), and acrylic (120 HU). Nineteen examples of the Test Object were manufactured. Initially, all Test Objects were scanned on the same CT scanner before the Test Objects were sent to the 20 specific sites and 42 individual CT scanners that were used in the study. The Test Objects were scanned over 17 months while the COPDGene study continued to recruit subjects. A mixed linear effect statistical analysis of the CT scans on the 19 Test Objects was performed. The statistical model reflected influence of reconstruction kernels, tube current, individual Test Objects, CT scanner models, and temporal consistency on CT attenuation. RESULTS: Depending on the Test Object material, there were significant differences between reconstruction kernels, tube current, individual Test Objects, CT scanner models, and temporal consistency. The two Test Object materials of most interest were lung equivalent foam and internal air. With lung equivalent foam, there were significant (p < 0.05) differences between the Siemens B31 (-856.6, ±0.82; mean ± SE) and the GE Standard (-856.6 ± 0.83) reconstruction kernel relative to the Siemens B35 reference standard (-852.5 ± 1.4). Comparing lung equivalent foam attenuation there were also significant differences between CT scanner models (p < 0.01), tube current (p < 0.005), and in temporal consistency (p < 0.005) at individual sites. However, there were no significant effects measurable using different examples of the Test Objects at the various sites compared to the reference scans of the 19 Test Objects. For internal air, significant (p < 0.005) differences were found between all reconstruction kernels (Siemens B31, GE Standard, and Phillips B) compared to the reference standard. There were significant differences between CT models (p < 0.005), and tube current (p < 0.005). There were no significant effects measurable using different examples of the Test Objects at the various sites compared to the reference scans of the 19 Test Objects. Differences, across scanners, between external air and internal air measures in this simple (relative to the in vivo lung) test object varied by as much as 15 HU. CONCLUSIONS: The authors conclude that the Test Object designed for this study was able to detect significant effects regarding individual CT scanners that altered the CT attenuation measurements relevant to the study that are used to determine lung density. Through an understanding of individual scanners, the Test Object analysis can be used to detect anomalies in an individual CT scanner and to statistically model out scanner differences and individual scanner changes over time in a large multicenter trial.

Machine learning identifies specific habitats associated with genetic connectivity in Hyla squirella.

The goal of this study was to identify and differentiate the influence of multiple habitat types that span a spectrum of suitability for Hyla squirella, a widespread frog species that occurs in a broad range of habitat types. We collected microsatellite data from 675 samples representing 20 localities from the southeastern USA and used machine-learning methodologies to identify significant habitat features associated with genetic structure. In simulation, we confirm that our machine-learning algorithm can successfully identify landscape features responsible for generating between-population genetic differentiation, suggesting that it can be a useful hypothesis-generating tool for landscape genetics. In our study system, we found that H. squirella were spatially structured and models including specific habitat types (i.e. upland oak forest and urbanization) consistently explained more variation in genetic distance (median pR(2) = 47.78) than spatial distance alone (median pR(2) = 23.81). Moreover, we estimate the relative importance that spatial distance, upland oak and urbanized habitat have in explaining genetic structure of H. squirella. We discuss how these habitat types may mechanistically facilitate dispersal in H. squirella. This study provides empirical support for the hypothesis that habitat-use can be an informative correlate of genetic differentiation, even for species that occur in a wide range of habitats.

Quantifying tissue heterogeneity using quadtree decomposition.

Volumetric computed tomography (CT) imaging provides a three-dimensional map of image intensities from which lung soft tissue density distribution can be estimated. The information gained from analyzing these images can prove valuable in diagnosis of conditions where lung tissue is damaged or has degenerated, and it is also necessary for modeling lung tissue mechanics. This paper presents a new technique for quantifying heterogeneity based on individual CT images, and investigates the heterogeneity of lung tissue in a group of healthy young subjects. It is intended that development of this technique leads to a standard model of classifying heterogeneity in lung tissue, while taking into account variables such as different imaging platforms and resolutions, and the position of the patient during imaging.

Subclinical atherosclerosis, airflow obstruction and emphysema: the MESA Lung Study.

Airflow obstruction is an independent risk factor for cardiovascular events in the general population. The affected vascular bed and contribution of emphysema to cardiovascular risk are unclear. We examined whether an obstructive pattern of spirometry and quantitatively defined emphysema were associated with subclinical atherosclerosis in the carotid, peripheral and coronary circulations. The Multi-Ethnic Study of Atherosclerosis recruited participants aged 45-84 yrs without clinical cardiovascular disease. Spirometry, carotid intima-media thickness (IMT), ankle-brachial index (ABI) and coronary artery calcium (CAC) were measured using standard protocols. Percentage of emphysema-like lung was measured in the lung windows of cardiac computed tomography scans among 3,642 participants. Multiple linear regression was used to adjust for cardiac risk factors, including C-reactive protein. Decrements in forced expiratory volume in 1 s (FEV(1)) and FEV(1)/forced vital capacity ratio were associated with greater internal carotid IMT, particularly among smokers (p=0.03 and p<0.001, respectively) whereas percentage emphysema was associated with reduced ABI regardless of smoking history (p=0.004). CAC was associated with neither lung function (prevalence ratio for the presence of CAC in severe airflow obstruction 0.99, 95% CI 0.91-1.07) nor percentage emphysema. An obstructive pattern of spirometry and emphysema were associated distinctly and independently with subclinical atherosclerosis in the carotid arteries and peripheral circulation, respectively, and were not independently related to CAC.

Exploration of the volumetric composition of human lung cancer nodules in correlated histopathology and computed tomography.

Gaining a complete and comprehensive understanding of lung cancer nodule histological compositions and how these tissues are represented in radiological data is important not only for expanding the current knowledge base of cancer growth and development but also has potential implications for classification standards, radiological diagnosis methods and for the evaluation of treatment response. In this study we generate large scale histological segmentations of the cancerous and non-cancerous tissues within resected lung nodules. We have implemented a processing pipeline which allows for the direct correlation between histological data and spatially corresponding computed tomography data. Utilizing these correlated datasets we evaluated the statistical separation between Hounsfield Unit (HU) histogram values for each tissue type. The findings of this study revealed that lung cancer nodules contain a complex intermixing of cellular tissue types and that trends exist in the relationship between these tissue types. It was found that the mean Hounsfield Unit values for isolated lung cancer nodules imaged with computed tomography, had statistically significantly different values for non-solid bronchoalveolar carcinoma, solid cancerous tumor, blood, and inactive fibrotic stromal tissue.

Protein profile of exosomes from trabecular meshwork cells.

To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular pressure control, the exosome proteome from primary cultures of human TM cell monolayers was analyzed. Exosomes were purified from urine and conditioned media from primary cultures of human TM cell monolayers and subjected to a two dimensional HPLC separation and MS/MS analyses using the MudPIT strategy. Spectra were searched against a human protein database using Sequest. Protein profiles were compared to each other and the Exocarta database and the presence of specific protein markers confirmed by Western blot analyses of exosomes from aqueous humor and human TM cell strains (n=5) that were untreated, or exposed to dexamethasone and/or ionomycin. TM cell exosomes contained 108 of the 143 most represented exosome proteins in ExoCarta, including previously characterized markers such as membrane organizing and tetraspanin proteins. Several cell-specific proteins in TM exosomes were identified including myocilin, emilin-1 and neuropilin-1. All TM exosome proteins had flotation densities on sucrose gradients and release responses to ionomycin typical for exosomes. Taken together, TM exosomes have a characteristic exosome protein profile plus contain unique proteins, including the glaucoma-causing protein, myocilin; suggesting a role for exosomes in the control of intraocular pressure.

The interdependent contributions of gravitational and structural features to perfusion distribution in a multiscale model of the pulmonary circulation.

Recent experimental and imaging studies suggest that the influence of gravity on the measured distribution of blood flow in the lung is largely through deformation of the parenchymal tissue. To study the contribution of hydrostatic effects to regional perfusion in the presence of tissue deformation, we have developed an anatomically structured computational model of the pulmonary circulation (arteries, capillaries, veins), coupled to a continuum model of tissue deformation, and including scale-appropriate fluid dynamics for blood flow in each vessel type. The model demonstrates that both structural and the multiple effects of gravity on the pulmonary circulation make a distinct contribution to the distribution of blood. It shows that postural differences in perfusion gradients can be explained by the combined effect of tissue deformation and extra-acinar blood vessel resistance to flow in the dependent tissue. However, gravitational perfusion gradients persist when the effect of tissue deformation is eliminated, highlighting the importance of the hydrostatic effects of gravity on blood distribution in the pulmonary circulation. Coupling of large- and small-scale models reveals variation in microcirculatory driving pressures within isogravitational planes due to extra-acinar vessel resistance. Variation in driving pressures is due to heterogeneous large-vessel resistance as a consequence of geometric asymmetry in the vascular trees and is amplified by the complex balance of pressures, distension, and flow at the microcirculatory level.

Food availability and sex reversal in Mytella charruana, an introduced bivalve in the southeastern United States.

We studied the reproductive biology of Mytella charruana to determine the potential reproductive success of this newly introduced bivalve species from Central/South America. We analyzed gonad morphology, gametogenesis, and the sex ratios of introduced populations throughout a 12 month period. In the non-native habitat M. charruana shows the same strategy of gametogenesis that had been observed in its native environment, which is an opportunistic type of gonadal cycle with gametes produced throughout the year. Instead, the spawning period of M. charruana along the southeastern US coast is extended compared to that found in the native environment. We determined the minimum size (shell length) of sexually reproductive mussels to be 1.25 cm. Interestingly, throughout the year the population samples were typically composed of a higher proportion of females. The female to male sex ratio varied within a wide range from 1:0 to 1:3.3. Upon this discovery we tested the effects of food availability on the gametogenesis of adult animals. The sex ratio of mussels collected from different locations and maintained in the laboratory with or without food changed toward a male-bias under starvation conditions within a month. This is the first study directly showing that food availability can trigger sex reversal in an adult bivalve. According to our data this mussel species will likely continue to spread along the east coast of the US. Moreover, M. charruana may prove to be a model organism in the study of alternative sexuality in bivalves.

Prevalence and clinical correlates of bronchoreversibility in severe emphysema.

Chronic obstructive pulmonary disease (COPD) exhibits airflow obstruction that is not fully reversible. The importance of bronchoreversibility remains controversial. We hypothesised that an emphysematous phenotype of COPD would be associated with decreased bronchoreversibility. 544 patients randomised to the medical arm of the National Emphysema Treatment Trial formed the study group. Participants underwent multiple measurements of bronchoreversibility on a mean of four sessions over 1.91 yrs. They were also characterised by measures of symptoms, quality of life and quantitative measures of emphysema by computed tomography. Mean baseline forced expiratory volume in 1 s (FEV(1)) in this patient population is 24% predicted. 22.2% of patients demonstrated bronchoreversibility on one or more occasions using American Thoracic Society/European Respiratory Society criteria. Few patients (0.37%) had bronchoreversibility on all completed tests. Patients who demonstrated bronchoreversibility were more likely to be male, and have better lung function and less emphysema. 64% of patients demonstrated large (> or =400 mL) changes in forced vital capacity (FVC). In a severe emphysema population, bronchoreversibility as defined by change in FEV(1) is infrequent, varies over time, and is more common in males and those with less severe emphysema. Improvements in FVC, however, were demonstrated in the majority of patients.

Species-specific pulmonary arterial asymmetry determines species differences in regional pulmonary perfusion.

The functional significance of differences in pulmonary vascular branching and diameter asymmetry between the human and quadruped lung has not previously been addressed. To evaluate the contribution of branching asymmetry to observable species differences in blood flow gradients, computed distributions of blood flow were compared in structure-based models of the human and ovine pulmonary arteries. The models were derived using a combination of computed tomography and a volume-filling algorithm. Pressure, flow, and deformed vessel diameter were calculated in both species models using equations representing conservation of mass and momentum, and a pressure-diameter relationship. The major difference between the human and ovine results was the presence of a large region of "zone 4" flow and higher mean flows in the central region of the ovine lung compared to that in the human. Heterogeneity in tissue perfusion and the contribution of gravity were similar in both species models; however, the gravitationally directed gradients of perfusion in the human and ovine models were different and each consistent with human and quadruped measurements, respectively. The results suggest that measured species differences in pulmonary perfusion gradients are largely determined by differences in branching asymmetry.

Role of aquaporin-1 in trabecular meshwork cell homeostasis during mechanical strain.

Aquaporin-1 (AQP1) channels are expressed by trabecular meshwork (TM) and Schlemm's canal cells of the conventional outflow pathway where fluid movement is predominantly paracellular, suggesting a non-canonical role for AQP1. We hypothesized that AQP1 functions to protect TM cells during periods of mechanical strain. To test this idea, primary cultures of confluent human TM cells on Bioflex membranes were exposed to static and cyclic stretch for 8 and 24h using the Flexcell system. AQP1 expression in TM cells was assessed by SDS-PAGE and Western blot using anti-AQP1 IgGs. AQP1 protein bands were analyzed using densitometry and normalized to beta-actin expression. Cell damage was monitored by measuring lactate dehydrogenase (LDH) and histone deacetylase appearance in conditioned media. Recombinant expression of AQP1 in TM cell cultures was facilitated by transduction with adenovirus. Results show that AQP1 expression significantly increased 2-fold with 10% static stretch and 3.5-fold with 20% static stretch at 8h (n=4, p<0.05) and 24h (n=6, p<0.05). While histone deacetylase levels were unaffected by treatments, release of LDH from TM cells was the most profound at the 20% static stretch level (n=4, p<0.05). Significantly, cells were refractory to the 20% static stretch level when AQP1 expression was increased to near tissue levels. Analysis of LDH release with respect to AQP1 expression revealed an inverse linear relationship (r(2)=0.7780). Taken together, AQP1 in human TM appears to serve a protective role by facilitating improved cell viability during conditions of mechanical strain.

Rapid prototyping raw models on the basis of high resolution computed tomography lung data for respiratory flow dynamics.

RATIONALE AND OBJECTIVES: Three-dimensional image reconstruction by volume rendering and rapid prototyping has made it possible to visualize anatomic structures in three dimensions for interventional planning and academic research. METHODS: Volumetric chest computed tomography was performed on a healthy volunteer. Computed tomographic images of the larger bronchial branches were segmented by an extended three-dimensional region-growing algorithm, converted into a stereolithography file, and used for computer-aided design on a laser sintering machine. The injection of gases for respiratory flow modeling and measurements using magnetic resonance imaging were done on a hollow cast. RESULTS: Manufacturing the rapid prototype took about 40 minutes and included the airway tree from trackea to segmental bronchi (fifth generation). The branching of the airways are clearly visible in the (3)He images, and the radial imaging has the potential to elucidate the airway dimensions. CONCLUSION: The results for flow patterns in the human bronchial tree using the rapid-prototype model with hyperpolarized helium-3 magnetic resonance imaging show the value of this model for flow phantom studies.

Permanent Genetic Resources added to Molecular Ecology Resources database 1 January 2009-30 April 2009.

This article documents the addition of 283 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Agalinis acuta; Ambrosia artemisiifolia; Berula erecta; Casuarius casuarius; Cercospora zeae-maydis; Chorthippus parallelus; Conyza canadensis; Cotesia sesamiae; Epinephelus acanthistius; Ficedula hypoleuca; Grindelia hirsutula; Guadua angustifolia; Leucadendron rubrum; Maritrema novaezealandensis; Meretrix meretrix; Nilaparvata lugens; Oxyeleotris marmoratus; Phoxinus neogaeus; Pristomyrmex punctatus; Pseudobagrus brevicorpus; Seiridium cardinale; Stenopsyche marmorata; Tetranychus evansi and Xerus inauris. These loci were cross-tested on the following species: Agalinis decemloba; Agalinis tenella; Agalinis obtusifolia; Agalinis setacea; Agalinis skinneriana; Cercospora zeina; Cercospora kikuchii; Cercospora sorghi; Mycosphaerella graminicola; Setosphaeria turcica; Magnaporthe oryzae; Cotesia flavipes; Cotesia marginiventris; Grindelia Xpaludosa; Grindelia chiloensis; Grindelia fastigiata; Grindelia lanceolata; Grindelia squarrosa; Leucadendron coniferum; Leucadendron salicifolium; Leucadendron tinctum; Leucadendron meridianum; Laodelphax striatellus; Sogatella furcifera; Phoxinus eos; Phoxinus rigidus; Phoxinus brevispinosus; Phoxinus bicolor; Tetranychus urticae; Tetranychus turkestani; Tetranychus ludeni; Tetranychus neocaledonicus; Tetranychus amicus; Amphitetranychus viennensis; Eotetranychus rubiphilus; Eotetranychus tiliarium; Oligonychus perseae; Panonychus citri; Bryobia rubrioculus; Schizonobia bundi; Petrobia harti; Xerus princeps; Spermophilus tridecemlineatus and Sciurus carolinensis.

Myocilin-associated exosomes in human ocular samples.

Mutations in myocilin result in ocular hypertension, likely due to decreased drainage of aqueous humor through the trabecular meshwork. Since less myocilin is found in the aqueous humor of those with disease-causing mutations, understanding myocilin's role in the aqueous humor is of clinical importance. Recently, myocilin was shown to exit cultured trabecular meshwork cells in association with shed vesicles called exosomes. To examine relevance of this finding in a physiological setting, the present study examined three different types of ocular samples for the presence of myocilin-associated exosomes. Using differential centrifugation steps, we found myocilin associated with exosomes isolated from effluent collected from human anterior segments in organ culture and aqueous humor obtained from human cadaveric eyes or from patients undergoing excisional surgery. Similar to results with cultured cells, myocilin associated predominately with exosomes in fresh samples, appeared mostly soluble at later times, and had biochemical properties (density of 1.13-1.19 g/ml in linear sucrose gradient) similar to those characteristics of exosomes. These data indicate that exosomes are present and may facilitate the transport of myocilin into the extracellular space of human ocular cells.

In vivo micro-CT lung imaging via a computer-controlled intermittent iso-pressure breath hold (IIBH) technique.

Respiratory research with mice using micro-computed tomography (micro-CT) has been predominantly hindered by the limited resolution and signal-to-noise ratio (SNR) as a result of respiratory motion artefacts. In this study, we develop a novel technique for capturing the lung microstructure in vivo using micro-CT, through a computer-controlled intermittent iso-pressure breath hold (IIBH), to reduce respiratory motion, increasing resolution and SNR of reconstructed images. We compare four gating techniques, i.e. no gating, late expiratory (LE) gating, late inspiratory (LI) gating and finally intermittent iso-pressure breath hold (IIBH) gating. Quantitatively, we compare several common image analysis methods used to extract valuable physiologic and anatomic information from the respiratory system, and show that the IIBH technique produces the most representative and repeatable results.

Coiled-coil targeting of myocilin to intracellular membranes.

Mutations in myocilin (MYOC) associate with glaucoma and ocular hypertension. Unfortunately, the specific role of MYOC, a widely expressed protein of unknown function, in ocular hypertension is unknown. Since MYOC localizes both to intracellular membranes and to the cytosol, we tested the hypothesis that MYOC is a cytosolic protein that associates with cellular membranes via its coiled-coil domain. Using green fluorescent protein (GFP) chimeras in expression and metabolic labeling studies, we observed that MYOC's putative signal peptide failed to traffic GFP into the secretory machinery and out of transfected cells. Next, we tested which of MYOC's three folding domains were responsible for targeting. In cell fractionation and immunofluorescence microscopy studies, the coiled-coil, but not the helix-turn-helix or olfactomedin domains, was necessary and sufficient to target GFP chimeras to cell membranes. Interestingly, a vesicular phenotype required sequential addition of the helix-turn-helix and olfactomedin domains to the coiled-coil. Taken together, these data indicate that the coiled-coil domain, not the putative signal sequence, is responsible for the targeting of MYOC to the secretory machinery.