Development of physiologically-informed computational coronary artery plaques for use in virtual imaging trials.

BACKGROUND: As a leading cause of death, worldwide, cardiovascular disease is of great clinical importance. Among cardiovascular diseases, coronary artery disease (CAD) is a key contributor, and it is the attributed cause of death for 10% of all deaths annually. The prevalence of CAD is commensurate with the rise in new medical imaging technologies intended to aid in its diagnosis and treatment. The necessary clinical trials required to validate and optimize these technologies require a large cohort of carefully controlled patients, considerable time to complete, and can be prohibitively expensive. A safer, faster, less expensive alternative is using virtual imaging trials (VITs), utilizing virtual patients or phantoms combined with accurate computer models of imaging devices. PURPOSE: In this work, we develop realistic, physiologically-informed models for coronary plaques for application in cardiac imaging VITs. METHODS: Histology images of plaques at micron-level resolution were used to train a deep convolutional generative adversarial network (DC-GAN) to create a library of anatomically variable plaque models with clinical anatomical realism. The stability of each plaque was evaluated by finite element analysis (FEA) in which plaque components and vessels were meshed as volumes, modeled as specialized tissues, and subjected to the range of normal coronary blood pressures. To demonstrate the utility of the plaque models, we combined them with the whole-body XCAT computational phantom to perform initial simulations comparing standard energy-integrating detector (EID) CT with photon-counting detector (PCD) CT. RESULTS: Our results show the network is capable of generating realistic, anatomically variable plaques. Our simulation results provide an initial demonstration of the utility of the generated plaque models as targets to compare different imaging devices. CONCLUSIONS: Vast, realistic, and variable CAD pathologies can be generated to incorporate into computational phantoms for VITs. There they can serve as a known truth from which to optimize and evaluate cardiac imaging technologies quantitatively.

Surface-based anthropomorphic bone structures for use in high-resolution simulated medical imaging.

Objective.Virtual imaging trials enable efficient assessment and optimization of medical image devices and techniques via simulation rather than physical studies. These studies require realistic, detailed ground-truth models or phantoms of the relevant anatomy or physiology. Anatomical structures within computational phantoms are typically based on medical imaging data; however, for small and intricate structures (e.g. trabecular bone), it is not reasonable to use existing clinical data as the spatial resolution of the scans is insufficient. In this study, we develop a mathematical method to generate arbitrary-resolution bone structures within virtual patient models (XCAT phantoms) to model the appearance of CT-imaged trabecular bone.Approach. Given surface definitions of a bone, an algorithm was implemented to generate stochastic bicontinuous microstructures to form a network to define the trabecular bone structure with geometric and topological properties indicative of the bone. For an example adult male XCAT phantom (50th percentile in height and weight), the method was used to generate the trabecular structure of 46 chest bones. The produced models were validated in comparison with published properties of bones. The utility of the method was demonstrated with pilot CT and photon-counting CT simulations performed using the accurate DukeSim CT simulator on the XCAT phantom containing the detailed bone models.Main results. The method successfully generated the inner trabecular structure for the different bones of the chest, having quantiative measures similar to published values. The pilot simulations showed the ability of photon-counting CT to better resolve the trabecular detail emphasizing the necessity for high-resolution bone models.Significance.As demonstrated, the developed tools have great potential to provide ground truth simulations to access the ability of existing and emerging CT imaging technology to provide quantitative information about bone structures.

A systematic assessment of photon-counting CT for bone mineral density and microarchitecture quantifications.

Photon-counting CT (PCCT) is an emerging imaging technology with potential improvements in quantification and rendition of micro-structures due to its smaller detector sizes. The aim of this study was to assess the performance of a new PCCT scanner (NAEOTOM Alpha, Siemens) in quantifying clinically relevant bone imaging biomarkers for characterization of common bone diseases. We evaluated the ability of PCCT in quantifying microarchitecture in bones compared to conventional energy-integrating CT. The quantifications were done through virtual imaging trials, using a 50 percentile BMI male virtual patient, with a detailed model of trabecular bone with varied bone densities in the lumbar spine. The virtual patient was imaged using a validated CT simulator (DukeSim) at CTDIvol of 20 and 40 mGy for three scan modes: ultra-high-resolution PCCT (UHR-PCCT), high-resolution PCCT (HR-PCCT), and a conventional energy-integrating CT (EICT) (FORCE, Siemens). Further, each scan mode was reconstructed with varying parameters to evaluate their effect on quantification. Bone mineral density (BMD), trabecular volume to total bone volume (BV/TV), and radiomics texture features were calculated in each vertebra. The most accurate BMD measurements relative to the ground truth were UHR-PCCT images (error: 3.3% ± 1.5%), compared to HR-PCCT (error: 5.3% ± 2.0%) and EICT (error: 7.1% ± 2.0%). UHR-PCCT images outperformed EICT and HR-PCCT. In BV/TV quantifications, UHR-PCCT (errors of 29.7% ± 11.8%) outperformed HR-PCCT (error: 80.6% ± 31.4%) and EICT (error: 67.3% ± 64.3). UHR-PCCT and HR-PCCT texture features were sensitive to anatomical changes using the sharpest kernel. Conversely, the texture radiomics showed no clear trend to reflect the progression of the disease in EICT. This study demonstrated the potential utility of PCCT technology in improved performance of bone quantifications leading to more accurate characterization of bone diseases.

The Behavioural Dysfunction Questionnaire discriminates behavioural variant frontotemporal dementia from Alzheimer's disease dementia and major depressive disorder.

BACKGROUND AND OBJECTIVES: Early-stage behavioural variant frontotemporal dementia (bvFTD) is often misdiagnosed, highlighting the need for new diagnostic instruments. Based on the revised diagnostic criteria for bvFTD, we developed the Behavioural Dysfunction Questionnaire (BDQ). In this explorative study, we aimed to determine the best scoring and analytical method for the BDQ to discriminate between bvFTD and non-bvFTD patients. MATERIALS AND METHODS: 34 patients with early-stage bvFTD, 56 with early-stage Alzheimer's disease dementia (ADD) and 41 with major depressive disorder (MDD) were recruited. We calculated BDQ-items with or without inclusion of a time criterion: (a) without time criterion, (b) with 10 years' time criterion (symptom presence less than 10 years), and (c) with 3 years' time criterion (symptom presentation within the first 3 years). Using these three differently calculated items, we generated six variables, i.e. 3*2 [BDQ-Global Score (BDQ-GS; domains average score); BDQ-Global Domain Score (BDQ-GDS; domains categorical score)]. Then, we performed univariate and bivariate (BDQ-GS and BDQ-GDS combined) ROC analyses. RESULTS: Models including BDQ-GS, BDQ-GDS or both variables combined discriminated similarly between groups. In contrast, models without time criterion or with 10 years' time criterion discriminated better than models including variables with 3 years' time criterion. These models discriminated highly (AUC = 85.98-87.78) between bvFTD and MDD and bvFTD and ADD, respectively. CONCLUSION: BDQ-scores without any time criterion discriminated highly between early-stage bvFTD and non-bvFTD groups, which could improve the early diagnosis of bvFTD. With its standardised procedure, the BDQ is also appropriate for repeated assessments.

Development and CT image-domain validation of a computational lung lesion model for use in virtual imaging trials.

PURPOSE: Computational abnormalities (e.g., lesion models) for use in medical imaging simulation studies are frequently generated using data collected from clinical images. Although this approach allows for highly-customizable lesion detectability studies on clinical computed tomography (CT) data, the ground-truth lesion models produced with this method do not provide a sufficiently realistic lesion morphology for use with current anthropomorphic simulation studies. This work is intended to demonstrate that the new anatomically-informed lesion model presented here is not inferior to the previous lesion model under CT imaging, and can therefore provide a more biologically-informed model for use with simulated CT imaging studies. METHODS: The lesion model was simulated initially from a seed cell with 10 µm diameter placed in an anatomical location within segmented lung CT and was allowed to reproduce locally within the available solid angle in discrete time-intervals (corresponding to synchronous cell cycles) up to a size of ∼200 µm in diameter. Daughter cells of generation G were allowed also to reproduce on the next available time-step given sufficient space. At lesion sizes beyond 200 µm in diameter, the health of subregions of cells were tracked with a Markov chain technique, indicating which regions were likely to continue growing, which were likely stable, and which were likely to develop necrosis given their proximity to anatomical features and other lesion cells. For lesion sizes beyond 500 µm, the lesion was represented with three nested, triangulated surfaces (corresponding to proliferating, dormant, and necrotic regions), indicating how discrete volumes of the lesion were behaving at a particular time. Lesions were then assigned smoothly-varying material properties based on their cellular level health in each region, resulting in a multi-material lesion model. The lesions produced with this model were then voxelized and placed into lung CT images for comparison with both prior work and clinical data. This model was subject to an observer study in which cardiothoracic imaging radiologists assessed the realism of both clinical and synthetic lesions in CT images. RESULTS: The useable outputs of this work were voxel- or surface-based, validated, computational lesions, at a scale clearly visible on clinical CT (3-4 cm). Analysis of the observer study results indicated that the computationally-generated lesions were indistinguishable from clinical lesions (AUC = 0.49, 95% CI = [0.36, 0.61]) and non-inferior to an earlier image-based lesion model-indicating the advantage of the model for use in both hybrid CT images and in simulated CT imaging of the lungs. CONCLUSIONS: Results indicated the non-inferiority of this model as compared to previous methods, indicating the utility of the model for use in both hybrid CT images and in simulated CT imaging.

Can Photon-Counting CT Improve Estimation Accuracy of Morphological Radiomics Features? A Simulation Study for Assessing the Quantitative Benefits from Improved Spatial Resolution in Deep Silicon-Based Photon-Counting CT.

RATIONALE AND OBJECTIVES: Deep silicon-based photon-counting CT (Si-PCCT) is an emerging detector technology that provides improved spatial resolution by virtue of its reduced pixel sizes. This article reports the outcomes of the first simulation study evaluating the impact of this advantage over energy-integrating CT (ECT) for estimation of morphological radiomics features in lung lesions. MATERIALS AND METHODS: A dynamic nutrient-access-based stochastic model was utilized to generate three distinct morphologies for lung lesions. The lesions were inserted into the lung parenchyma of an anthropomorphic phantom (XCAT - 50th percentile BMI) at 50, 70, and 90 mm from isocenter. The phantom was virtually imaged with an imaging simulator (DukeSim) modeling a Si-PCCT and a conventional ECT system using varying imaging conditions (dose, reconstruction kernel, and pixel size). The imaged lesions were segmented using a commercial segmentation tool (AutoContour, Advantage Workstation Server 3.2, GE Healthcare) followed by extraction of morphological radiomics features using an open-source radiomics package (pyradiomics). The estimation errors for both systems were computed as percent differences from corresponding feature values estimated for the ground-truth lesions. RESULTS: Compared to ECT, the mean estimation error was lower for Si-PCCT (independent features: 35.9% vs. 54.0%, all features: 54.5% vs. 68.1%) with statistically significant reductions in errors for 8/14 features. For both systems, the estimation accuracy was minimally affected by dose and distance from the isocenter while reconstruction kernel and pixel size were observed to have a relatively stronger effect. CONCLUSION: For all lesions and imaging conditions considered, Si-PCCT exhibited improved estimation accuracy for morphological radiomics features over a conventional ECT system, demonstrating the potential of this technology for improved quantitative imaging.

Anatomically and physiologically informed computational model of hepatic contrast perfusion for virtual imaging trials.

PURPOSE: Virtual (in silico) imaging trials (VITs), involving computerized phantoms and models of the imaging process, provide a modern alternative to clinical imaging trials. VITs are faster, safer, and enable otherwise-impossible investigations. Current phantoms used in VITs are limited in their ability to model functional behavior such as contrast perfusion which is an important determinant of dose and image quality in CT imaging. In our prior work with the XCAT computational phantoms, we determined and modeled inter-organ (organ to organ) intravenous contrast concentration as a function of time from injection. However, intra-organ concentration, heterogeneous distribution within a given organ, was not pursued. We extend our methods in this work to model intra-organ concentration within the XCAT phantom with a specific focus on the liver. METHODS: Intra-organ contrast perfusion depends on the organ's vessel network. We modeled the intricate vascular structures of the liver, informed by empirical and theoretical observations of anatomy and physiology. The developed vessel generation algorithm modeled a dual-input-single-output vascular network as a series of bifurcating surfaces to optimally deliver flow within the bounding surface of a given XCAT liver. Using this network, contrast perfusion was simulated within voxelized versions of the phantom by using knowledge of the blood velocities in each vascular structure, vessel diameters and length, and the time since the contrast entered the hepatic artery. The utility of the enhanced phantom was demonstrated through a simulation study with the phantom voxelized prior to CT simulation with the relevant liver vasculature prepared to represent blood and iodinated contrast media. The spatial extent of the blood-contrast mixture was compared to clinical data. RESULTS: The vascular structures of the liver were generated with size and orientation which resulted in minimal energy expenditure required to maintain blood flow. Intravenous contrast was simulated as having known concentration and known total volume in the liver as calibrated from time-concentration curves. Measurements of simulated CT ROIs were found to agree with clinically observed values of early arterial phase contrast enhancement of the parenchyma ( ∼ 5 $ \sim 5$ HU). Similarly, early enhancement in the hepatic artery was found to agree with average clinical enhancement ( 180 $(180$ HU). CONCLUSIONS: The computational methods presented here furthered the development of the XCAT phantoms allowing for multi-timepoint contrast perfusion simulations, enabling more anthropomorphic virtual clinical trials intended for optimization of current clinical imaging technologies and applications.

Development and validation of an automated methodology to assess perceptual in vivo noise texture in liver CT.

Purpose: Developing, validating, and evaluating a method for measuring noise texture directly from patient liver CT images (i.e., in vivo). Approach: The method identifies target regions within patient scans that are least likely to have major contribution of patient anatomy, detrends them locally, and measures noise power spectrum (NPS) there using a previously phantom-validated technique targeting perceptual noise-non-anatomical fluctuations in the image that may interfere with the detection of focal lesions. Method development and validation used scanner-specific CT simulations of computational, anthropomorphic phantom (XCAT phantom, three phases of contrast-enhancement) with known ground truth of the NPS. Simulations were based on a clinical scanner (Definition Flash, Siemens) and clinically relevant settings (tube voltage of 120 kV at three dose levels). Images were reconstructed with filtered backprojection (kernel: B31, B41, and B50) and Sinogram Affirmed Iterative Reconstruction (kernel: I31, I41, and I50) using a manufacturer-specific reconstruction software (ReconCT, Siemens). All NPS measurements were made in the liver. Ground-truth NPS were taken as the sum of (1) a measurement in parenchymal regions of anatomy-subtracted (i.e., noise only) scans, and (2) a measurement in the same region of noise-free (pre-noise-insertion) images. To assess in vivo NPS performance, correlation of NPS average frequency ( f avg ), was reported. Sensitivity of accuracy [root-mean-square-error (RMSE)] to number of pixels included in measurement was conducted via bootstrapped pixel-dropout. Sensitivity of NPS to dose and reconstruction kernel was assessed to confirm that ground truth NPS similarities were maintained in patient-specific measurements. Results: Pearson and Spearman correlation coefficients 0.97 and 0.96 for f avg indicated good correlation. Results suggested accurate NPS measurements (within 5% total RMSE) could be acquired with ∼ 10 6 pixels . Conclusions: Relationships of similar NPS due to reconstruction kernel and dose were preserved between gold standard and observed in vivo estimations. The NPS estimation method was further deployed on clinical cases to demonstrate the feasibility of clinical analysis.

Quantification of Minimum Detectable Difference in Radiomics Features Across Lesions and CT Imaging Conditions.

RATIONALE AND OBJECTIVES: The 3-fold purpose of this study was to (1) develop a method to relate measured differences in radiomics features in different computed tomography (CT) scans to one another and to true feature differences; (2) quantify minimum detectable change in radiomics features based on measured radiomics features from pairs of synthesized CT images acquired under variable CT scan settings, and (3) ascertain and inform the recommendations of the Quantitative Imaging Biomarkers Alliance (QIBA) for nodule volumetry. MATERIALS AND METHODS: Images of anthropomorphic lung nodule models were simulated using resolution and noise properties for 297 unique imaging conditions. Nineteen morphology features were calculated from both the segmentation masks derived from the imaged nodules and from ground truth nodules. Analysis was performed to calculate minimum detectable difference of radiomics features as a function of imaging protocols in comparison to QIBA guidelines. RESULTS: The minimum detectable differences ranged from 1% to 175% depending on the specific feature and set of imaging protocols. The results showed that QIBA protocol recommendations result in improved minimum detectable difference as compared to the range of possible protocols. The results showed that the minimum detectable differences may be improved from QIBA's current recommendation by further restricting the slice thickness requirement to be between 0.5 mm and 1 mm. CONCLUSION: Minimum detectable differences of radiomics features were quantified for lung nodules across a wide range of possible protocols. The results can be used prospectively to inform decision-making about imaging protocols to provide superior quantification of radiomics features.

Three-dimensional regions-of-interest-based intra-operative four-dimensional soft tissue perfusion imaging using a standard x-ray system with no gantry rotation: A simulation study for a proof of concept.

PURPOSE: Many interventional procedures aim at changing soft tissue perfusion or blood flow. One problem at present is that soft tissue perfusion and its changes cannot be assessed in an interventional suite because cone-beam computed tomography is too slow (it takes 4-10 s per volume scan). In order to address the problem, we propose a novel method called IPEN for Intra-operative four-dimensional soft tissue PErfusion using a standard x-ray system with No gantry rotation. METHODS: IPEN uses two input datasets: (a) the contours and locations of three-dimensional regions-of-interest (ROIs) such as arteries and sub-sections of cancerous lesions, and (b) a series of x-ray projection data obtained from an intra-arterial contrast injection to contrast enhancement to wash-out. IPEN then estimates a time-enhancement curve (TEC) for each ROI directly from projections without reconstructing cross-sectional images by maximizing the agreement between synthesized and measured projections with a temporal roughness penalty. When path lengths through ROIs are known for each x-ray beam, the ROI-specific enhancement can be accurately estimated from projections. Computer simulations are performed to assess the performance of the IPEN algorithm. Intra-arterial contrast-enhanced liver scans over 25 s were simulated using XCAT phantom version 2.0 with heterogeneous tissue textures and cancerous lesions. The following four sub-studies were performed: (a) The accuracy of the estimated TECs with overlapped lesions was evaluated at various noise (dose) levels with either homogeneous or heterogeneous lesion enhancement patterns; (b) the accuracy of IPEN with inaccurate ROI contours was assessed; (c) we investigated how overlapping ROIs and noise in projections affected the accuracy of the IPEN algorithm; and (d) the accuracy of the perfusion indices was assessed. RESULTS: The TECs estimated by IPEN were sufficiently accurate at a reference dose level with the root-mean-square deviation (RMSD) of 0.0027 ± 0.0001 cm-1 or 13 ± 1 Hounsfield unit (mean ± standard deviation) for the homogeneous lesion enhancement and 0.0032 ± 0.0005 cm-1 for the heterogeneous enhancement (N = 20 each). The accuracy was degraded with decreasing doses: The RMSD with homogeneous enhancement was 0.0220 ± 0.0003 cm-1 for 20% of the reference dose level. Performing 3 × 3 pixel averaging on projection data improved the RMSDs to 0.0051 ± 0.0002 cm-1 for 20% dose. When the ROI contours were inaccurate, smaller ROI contours resulted in positive biases in TECs, whereas larger ROI contours produced negative biases. The bias remained small, within ± 0.0070 cm-1 , when the Sorenson-Dice coefficients (SDCs) were larger than 0.81. The RMSD of the TEC estimation was strongly associated with the condition of the problem, which can be empirically quantified using the condition number of a matrix A z that maps a vector of ROI enhancement values z to projection data and a weighted variance of projection data: a linear correlation coefficient (R) was 0.794 (P < 0.001). The perfusion index values computed from the estimated TECs agreed well with the true values (R ≥ 0.985, P < 0.0001). CONCLUSION: The IPEN algorithm can estimate ROI-specific TECs with high accuracy especially when 3 × 3 pixel averaging is applied, even when lesion enhancement is heterogeneous, or ROI contours are inaccurate but the SDC is at least 0.81.

Lysophosphatidic acid-induced pro-thrombotic phosphatidylserine exposure and ionophore-induced microvesiculation is mediated by the scramblase TMEM16F in erythrocytes.

Recent studies indicate that erythrocytes actively modulate blood clotting and thrombus formation. The lipid mediator lysophosphatidic acid (LPA) is produced by activated platelets, and triggers a signaling process in erythrocytes. This results in cellular calcium uptake and exposure of phosphatidylserine (PS) at the cell surface, thereby generating activated membrane binding sites for factors of the clotting cascade. Moreover, erythrocytes of patients with a bleeding disorder and mutations in the scramblase TMEM16F show impaired PS exposure and microvesiculation upon treatment with calcium ionophore. We report that TMEM16F inhibitors tannic acid (TA) and epigallocatechin-3-gallate (EGCG) inhibit LPA-induced PS exposure and calcium uptake at low micromolar concentrations; fluoxetine, an antidepressant and a known activator of TMEM16F, enhances these processes. These effectors likewise modulate erythrocyte PS exposure and microvesicle shedding induced by calcium ionophore treatment. Further, LPA-treated erythrocytes triggered thrombin generation in platelet-free plasma which was partially impaired in the presence of TA and EGCG. Thus, this study suggests that LPA activates the scramblase TMEM16F in erythrocytes, thereby possibly mediating a pro-thrombotic function in these cells. EGCG as well as fluoxetine, substances with potentially high plasma concentrations due to alimentation or medical treatment, should be considered as potential effectors of systemic hemostatic regulation.

Systematic analysis of bias and variability of morphologic features for lung lesions in computed tomography.

We propose to characterize the bias and variability of quantitative morphology features of lung lesions across a range of computed tomography (CT) imaging conditions. A total of 15 lung lesions were simulated (five in each of three spiculation classes: low, medium, and high). For each lesion, a series of simulated CT images representing different imaging conditions were synthesized by applying three-dimensional blur and adding correlated noise based on the measured noise and resolution properties of five commercial multislice CT systems, representing three dose levels ( CTDI vol of 1.90, 3.75, 7.50 mGy), three slice thicknesses (0.625, 1.25, 2.5 mm), and 33 clinical reconstruction kernels from five clinical scanners. The images were segmented using three segmentation algorithms and each algorithm was evaluated by computing a Sørensen-Dice coefficient between the ground truth and the segmentation. A series of 21 shape-based morphology features were extracted from both "ground truth" (i.e., preblur without noise) and "image rendered" lesions (i.e., postblur and with noise). For each morphology feature, the bias was quantified by comparing the percentage relative error in the morphology metric between the imaged lesions and the ground-truth lesions. The variability was characterized by calculating the average coefficient of variation averaged across repeats and imaging conditions. The active contour segmentation had the highest average Dice coefficient of 0.80 followed by 0.63 for threshold, and 0.39 for fuzzy c-means. The bias of the features was segmentation algorithm and feature-dependent, with sharper kernels being less biased and smoother kernels being more biased in general. The feature variability from simulated images ranged from 0.30% to 10% for repeats of the same condition and from 0.74% to 25.3% for different lesions in the same spiculation class. In conclusion, the bias of morphology features is dependent on the acquisition protocol in combination with the segmentation algorithm used and the variability is primarily dependent on the segmentation algorithm.

Odorous compounds sources and transport from a swine deep-pit finishing operation: A case study.

Odor emissions from swine finishing operations are an air quality issue that affects residents at the local level. A study was conducted at a commercial swine deep-pit finishing operation in central Iowa to monitor odorous compounds emitted and transported offsite. Gaseous compounds were sampled using either sorbent tubes or canisters with GC/MS analysis, and particulates matter (PM10) were sampled with high volume samplers and thermally extracted onto sorbent tubes for GC/MS analysis. Major odorous chemical classes detected at the swine facility included volatile sulfur compounds (VSC), volatile fatty acids (VFA), phenol and indole compounds. Manure storage was the main source of odorous compounds of which hydrogen sulfide (H2S), methanethiol, 4-methylphenol, and 3-methylindole were key offenders. Only H2S and 4-methylphenol were detected above odor threshold values (OTV) at all locations around the facility and both 4-methylphenol and 3-methylindole were detected above their OTV 1.5 km downwind from the swine facility. Odorous compounds generated during agitation and pumping of the deep pits was mainly H2S. Odorants were mainly transported in the gas phase with less than 0.1% being associated with PM10. Odor mitigation efforts should focus on gaseous compounds emitted from deep-pits and especially during manure agitation and deep-pit pumping.

Drug-induced endovesiculation of erythrocytes is modulated by the dynamics in the cytoskeleton/membrane interaction.

Recent studies on erythrocyte membrane fluctuations revealed that the erythrocyte cytoskeleton actively modulates its membrane association thereby regulating crucial membrane properties. Cationic amphiphilic drugs like chlorpromazine are known to induce a cup-like cell shape and vesicle formation into the cell interior, effectors of this process, however, are largely unknown. Using flow cytometry, this study explored conditions that influence endovesiculation induced by chlorpromazine. We found that inhibitors of membrane fluctuations, like ATP depletion, vanadate or fluoride, also inhibited endovesiculation whereas activation of PKC, known to decrease cytoskeleton association and increase membrane fluctuations, also enhanced endovesicle formation. This indicates that endovesicle formation and membrane fluctuations are modulated by the same cytoskeleton-regulated membrane properties. Further, acanthocytic erythrocytes of chorea acanthocytosis (ChAc) patients that lack the VPS13A/chorein protein - likely a crucial organizer at the erythrocyte cytoskeleton/membrane interface - showed a strong decrease in chlorpromazine-induced endovesiculation. The responses of ChAc erythrocytes to effectors of endovesiculation were similar to that of control erythrocytes, yet at drastically reduced levels. This suggests a more rigid and less dynamic interaction at the membrane-cytoskeleton interphase of ChAc erythrocytes.

An analogue-sensitive approach identifies basal body rotation and flagellum attachment zone elongation as key functions of PLK in Trypanosoma brucei.

Polo-like kinases are important regulators of cell division, playing diverse roles in mitosis and cytoskeletal inheritance. In the parasite Trypanosoma brucei, the single PLK homologue TbPLK is necessary for the assembly of a series of essential organelles that position and adhere the flagellum to the cell surface. Previous work relied on RNA interference or inhibitors of undefined specificity to inhibit TbPLK, both of which have significant experimental limitations. Here we use an analogue-sensitive approach to selectively and acutely inhibit TbPLK. T. brucei cells expressing only analogue-sensitive TbPLK (TbPLK(as)) grow normally, but upon treatment with inhibitor develop defects in flagellar attachment and cytokinesis. TbPLK cannot migrate effectively when inhibited and remains trapped in the posterior of the cell throughout the cell cycle. Using synchronized cells, we show that active TbPLK is a direct requirement for the assembly and extension of the flagellum attachment zone, which adheres the flagellum to the cell surface, and for the rotation of the duplicated basal bodies, which positions the new flagellum so that it can extend without impinging on the old flagellum. This approach should be applicable to the many kinases found in the T. brucei genome that lack an ascribed function.

Embryonic stem cells facilitate the isolation of persistent clonal cardiovascular progenitor cell lines and leukemia inhibitor factor maintains their self-renewal and myocardial differentiation potential in vitro.

Compelling evidence for the existence of somatic stem cells in the heart of different mammalian species has been provided by numerous groups; however, so far it has not been possible to maintain these cells as self-renewing and phenotypically stable clonal cell lines in vitro. Thus, we sought to identify a surrogate stem cell niche for the isolation and persistent maintenance of stable clonal cardiovascular progenitor cell lines, enabling us to study the mechanism of self-renewal and differentiation in these cells. Using postnatal murine hearts with a selectable marker as the stem cell source and embryonic stem cells and leukemia inhibitory factor (LIF)-secreting fibroblasts as a surrogate niche, we succeeded in the isolation of stable clonal cardiovascular progenitor cell lines. These cell lines self-renew in an LIF-dependent manner. They express both stemness transcription factors Oct4, Sox2, and Nanog and early myocardial transcription factors Nkx2.5, GATA4, and Isl-1 at the same time. Upon LIF deprivation, they exclusively differentiate to functional cardiomyocytes and endothelial and smooth muscle cells, suggesting that these cells are mesodermal intermediates already committed to the cardiogenic lineage. Cardiovascular progenitor cell lines can be maintained for at least 149 passages over 7 years without phenotypic changes, in the presence of LIF-secreting fibroblasts. Isolation of wild-type cardiovascular progenitor cell lines from adolescent and old mice has finally demonstrated the general feasibility of this strategy for the isolation of phenotypically stable somatic stem cell lines.

Tilt and decentration of an intraocular lens implanted in the ciliary sulcus after capsular bag defect during cataract surgery.

PURPOSE: To determine the impact of ciliary sulcus implantation after capsular bag defect on tilt and decentration of foldable intraocular lenses (IOL) measured by a novel device. METHODS: This prospective noncomparative single-center investigation included patients (N = 10) older than age 60 who underwent cataract surgery with a posterior capsular bag defect and implantation of a threepiece hydrophilic aberration neutral IOL (OphthalmoPro AC 7013) in the ciliary sulcus. All eyes (N = 10) were examined using the OPD-Scan II (Nidek). Spherical aberration (Z4,0), vertical coma (Z3,-1), and horizontal coma (Z3,1) were calculated for a 5.0 mm pupil. Lens tilt and decentration was studied using a new Purkinjemeter. RESULTS: The mean horizontal optic tilt was 7.68° ± 5.16 (SD) and the mean vertical optic tilt was 3.01° ± 2.44 (SD). Horizontal decentration was 0.4 ± 0.33 mm (SD) and vertical decentration was 0.31 ± 0.21 mm (SD). Corneal aberrations were partially compensated by IOL- related aberrations. CONCLUSIONS: Both tilt and decentration of sulcus-fixated IOLs exceeded the tolerable amounts evaluated for aspheric IOLs in eye model experiments. However, these spheric IOLs showed mitigation rather than accentuation of corneal wavefront aberrations in the rare event of capsular bag defect during cataract surgery.

Decentration and tilt of a single-piece aspheric intraocular lens compared with the lens position in young phakic eyes.

PURPOSE: To compare the tilt and decentration of a single-piece aspheric intraocular lens (IOL) and the position of the natural crystalline lens in young individuals. SETTING: Department of Ophthalmology, Knappschafts Hospital, Sulzbach, Germany. METHODS: This prospective noncomparative single-center study included volunteers younger than 40 years with no ocular abnormalities (crystalline lens group) and patients older than age 62 who had uneventful cataract surgery and Tecnis ZCB00 IOL implantation (IOL group). All eyes were examined with the OPD-Scan II; spherical aberration, vertical coma Z(3,-1), and horizontal coma Z(3,1) were analyzed with a 5.0 mm pupil. Lens tilt and decentration were studied with a new Purkinje meter. RESULTS: The crystalline lens group and IOL group comprised 20 subjects (40 eyes) each. Both groups showed compensation for corneal spherical aberration and neutralization of horizontal corneal coma. All lenses were tilted upward (mean 2.2 degrees, crystalline lens; 2.5 degrees, IOL) and to the temporal side (mean 3.1 degrees, crystalline lens; 2.6 degrees, IOL). The crystalline lenses were decentered downward (mean 0.16 mm) and to the temporal side (mean 0.07 mm). The IOL was displaced to the nasal side of the pupil (mean 0.06 mm) with almost no vertical mean decentration (0.02 mm upward). CONCLUSIONS: The aspheric IOL and young crystalline lens compensated for mean corneal spherical aberration, resulting in low total spherical aberration. The position of the IOLs showed minimal decentration and tilt and was mirror symmetrical, comparable to the position of the crystalline lens in young individuals. The slight malpositioning partially compensated for corneal horizontal coma.

Bone marrow cells are a rich source of growth factors and cytokines: implications for cell therapy trials after myocardial infarction.

AIMS: Results from clinical trials suggest that cardiac function after acute myocardial infarction (AMI) can be enhanced by an intracoronary infusion of autologous unselected nucleated bone marrow cells (BMCs). Release of paracrine factors has been proposed as a mechanism for these therapeutic effects; however, this hypothesis has not been tested in humans. METHODS AND RESULTS: BMCs and peripheral blood leucocytes (PBLs) were obtained from 15 patients with AMI and cultured in serum-free medium to obtain conditioned supernatants (SN). BMC-SN stimulated human coronary artery endothelial cell proliferation, migration, and tube formation, and induced cell sprouting in a mouse aortic ring assay. Moreover, BMC-SN protected rat cardiomyocytes from cell death induced by simulated ischaemia or ischaemia followed by reperfusion. While PBL-SN promoted similar effects on endothelial cells and cardiomyocytes, BMC-SN and PBL-SN in combination promoted synergistic effects. As shown by ProteinChip and GeneChip array analyses (each performed in triplicate), BMCs and PBLs expressed distinct patterns of pro-angiogenic and cytoprotective secreted factors. CONCLUSION: Our data support the paracrine hypothesis and suggest that characterization of the BMC secretome may lead to an identification of factors with therapeutic potential after AMI.

Evidence for a size-sensing mechanism in animal cells.

Continuously proliferating cells exactly double their mass during each cell cycle. Here we have addressed the controversial question of if and how cell size is sensed and regulated. We used erythroblasts that proliferate under the control of a constitutively active oncogene (v-ErbB) or under the control of physiological cytokines (stem cell factor, erythropoietin and v-ErbB inhibitor). The oncogene-driven cells proliferated 1.7 times faster and showed a 1.5-fold increase in cell volume. The two phenotypes could be converted into each other 24 h after altering growth factor signalling. The large cells had a higher rate of protein synthesis, together with a shortened G1 phase. Additional experiments with chicken erythroblasts and mouse fibroblasts, synchronized by centrifugal elutriation, provided further evidence that vertebrate cells can respond to cell size alterations (induced either through different growth factor signalling or DNA synthesis inhibitors) by compensatory shortening of the subsequent G1 phase. Taken together, these data suggest that an active size threshold mechanism exists in G1, which induces adjustment of cell-cycle length in the next cycle, thus ensuring maintenance of a proper balance between growth and proliferation rates in vertebrates.