Airway contraction and cytokine release in isolated rat lungs induced by wear particles from the road and tire interface and road vehicle brakes.

The dominant road traffic particle sources are wear particles from the road and tire interface, and from vehicle brake pads. The aim of this work was to investigate the effect of road and brake wear particles on pulmonary function and biomarkers in isolated perfused rat lungs. Particles were sampled from the studded tire wear of three road pavements containing different rock materials in a road simulator; and from the wear of two brake pad materials using a pin-on-disk machine. Isolated rat lungs inhaled the coarse and fine fractions of the sampled particles resulting in an estimated total particle lung dose of 50 µg. The tidal volume (TV) was measured during the particle exposure and the following 50 min. Perfusate and BALF were analyzed for the cytokines TNF, CXCL1 and CCL3. The TV of lungs exposed to rock materials was significantly reduced after 25 min of exposure compared to the controls, for quartzite already after 4 min. The particles of the heavy-duty brake pads had no effect on the TV. Brake particles resulted in a significant elevation of CXCL1 in the perfusate. Brake particles showed significant elevations of all three measured cytokines, and quartzite showed a significant elevation of TNF in BALF. The study shows that the toxic effect on lungs exposed to airborne particles can be investigated using measurements of tidal volume. Furthermore, the study shows that the choice of rock material in road pavements has the potential to affect the toxicity of road wear PM10.

Upper bounds on focusing efficiency.

Upper bounds on the focusing efficiency of aperture fields and lens systems are formulated using integral equation representations of Maxwell's equations and Lagrangian duality. Two forms of focusing efficiency are considered based on lens exit plane fields and optimal polarization currents within lens design regions of prescribed shape and available materials. Bounds are compared against the performance of classical prescriptions of ideal lens aperture fields, hyperbolic lens designs, and lenses produced by inverse design. Results demonstrate that, without regularization, focusing efficiency based solely on lens exit plane fields is unbounded, similar to the problem of unbounded antenna directivity. Additionally, results considering extruded two-dimensional dielectric geometries driven by out-of-plane electric fields for the calculation of bounds and inverse design demonstrate that aperture fields based on time-reversal do not necessarily yield optimal lens focusing efficiency, particularly in the case of near-field (high numerical aperture) focusing.

A Review of Road Traffic-Derived Non-Exhaust Particles: Emissions, Physicochemical Characteristics, Health Risks, and Mitigation Measures.

Implementation of regulatory standards has reduced exhaust emissions of particulate matter from road traffic substantially in the developed world. However, nonexhaust particle emissions arising from the wear of brakes, tires, and the road surface, together with the resuspension of road dust, are unregulated and exceed exhaust emissions in many jurisdictions. While knowledge of the sources of nonexhaust particles is fairly good, source-specific measurements of airborne concentrations are few, and studies of the toxicology and epidemiology do not give a clear picture of the health risk posed. This paper reviews the current state of knowledge, with a strong focus on health-related research, highlighting areas where further research is an essential prerequisite for developing focused policy responses to nonexhaust particles.

Fundamental bounds on the performance of monochromatic passive cloaks.

Fundamental bounds on the performance of monochromatic scattering-cancellation and field-zeroing cloaks made of prescribed linear passive materials occupying a predefined design region are formulated by projecting field quantities onto a sub-sectional basis and applying quadratically constrained quadratic programming. Formulations are numerically tested revealing key physical trends as well as advantages and disadvantages between the two classes of cloaks. Results show that the use of low-loss materials with high dielectric contrast affords the highest potential for effective cloaking.

Trade-offs in absorption and scattering by nanophotonic structures.

Trade-offs between absorption and scattering cross sections of lossy obstacles confined to an arbitrarily shaped volume are formulated as a multi-objective optimization problem solvable by Lagrangian-dual methods. Solutions to this optimization problem yield a Pareto-optimal set, the shape of which reveals the feasibility of achieving simultaneously extremal absorption and scattering. Two forms of the trade-off problems are considered involving both pre-assigned loss and reactive material parameters. Numerical comparisons between the derived multi-objective bounds and several classes of realized structures are made. Additionally, low-frequency (electrically small, long wavelength) limits are examined for certain special cases.

COMBImage2: a parallel computational framework for higher-order drug combination analysis that includes automated plate design, matched filter based object counting and temporal data mining.

BACKGROUND: Pharmacological treatment of complex diseases using more than two drugs is commonplace in the clinic due to better efficacy, decreased toxicity and reduced risk for developing resistance. However, many of these higher-order treatments have not undergone any detailed preceding in vitro evaluation that could support their therapeutic potential and reveal disease related insights. Despite the increased medical need for discovery and development of higher-order drug combinations, very few reports from systematic large-scale studies along this direction exist. A major reason is lack of computational tools that enable automated design and analysis of exhaustive drug combination experiments, where all possible subsets among a panel of pre-selected drugs have to be evaluated. RESULTS: Motivated by this, we developed COMBImage2, a parallel computational framework for higher-order drug combination analysis. COMBImage2 goes far beyond its predecessor COMBImage in many different ways. In particular, it offers automated 384-well plate design, as well as quality control that involves resampling statistics and inter-plate analyses. Moreover, it is equipped with a generic matched filter based object counting method that is currently designed for apoptotic-like cells. Furthermore, apart from higher-order synergy analyses, COMBImage2 introduces a novel data mining approach for identifying interesting temporal response patterns and disentangling higher- from lower- and single-drug effects. COMBImage2 was employed in the context of a small pilot study focused on the CUSP9v4 protocol, which is currently used in the clinic for treatment of recurrent glioblastoma. For the first time, all 246 possible combinations of order 4 or lower of the 9 single drugs consisting the CUSP9v4 cocktail, were evaluated on an in vitro clonal culture of glioma initiating cells. CONCLUSIONS: COMBImage2 is able to automatically design and robustly analyze exhaustive and in general higher-order drug combination experiments. Such a versatile video microscopy oriented framework is likely to enable, guide and accelerate systematic large-scale drug combination studies not only for cancer but also other diseases.

Optical theorems and physical bounds on absorption in lossy media.

Two different versions of an optical theorem for a scattering body embedded inside a lossy background medium are derived in this paper. The corresponding fundamental upper bounds on absorption are then obtained in closed form by elementary optimization techniques. The first version is formulated in terms of polarization currents (or equivalent currents) inside the scatterer and generalizes previous results given for a lossless medium. The corresponding bound is referred to here as a variational bound and is valid for an arbitrary geometry with a given material property. The second version is formulated in terms of the T-matrix parameters of an arbitrary linear scatterer circumscribed by a spherical volume and gives a new fundamental upper bound on the total absorption of an inclusion with an arbitrary material property (including general bianisotropic materials). The two bounds are fundamentally different as they are based on different assumptions regarding the structure and the material property. Numerical examples including homogeneous and layered (core-shell) spheres are given to demonstrate that the two bounds provide complimentary information in a given scattering problem.

COMBImage: a modular parallel processing framework for pairwise drug combination analysis that quantifies temporal changes in label-free video microscopy movies.

BACKGROUND: Large-scale pairwise drug combination analysis has lately gained momentum in drug discovery and development projects, mainly due to the employment of advanced experimental-computational pipelines. This is fortunate as drug combinations are often required for successful treatment of complex diseases. Furthermore, most new drugs cannot totally replace the current standard-of-care medication, but rather have to enter clinical use as add-on treatment. However, there is a clear deficiency of computational tools for label-free and temporal image-based drug combination analysis that go beyond the conventional but relatively uninformative end point measurements. RESULTS: COMBImage is a fast, modular and instrument independent computational framework for in vitro pairwise drug combination analysis that quantifies temporal changes in label-free video microscopy movies. Jointly with automated analyses of temporal changes in cell morphology and confluence, it performs and displays conventional cell viability and synergy end point analyses. The image processing algorithms are parallelized using Google's MapReduce programming model and optimized with respect to method-specific tuning parameters. COMBImage is shown to process time-lapse microscopy movies from 384-well plates within minutes on a single quad core personal computer. This framework was employed in the context of an ongoing drug discovery and development project focused on glioblastoma multiforme; the most deadly form of brain cancer. Interesting add-on effects of two investigational cytotoxic compounds when combined with vorinostat were revealed on recently established clonal cultures of glioma-initiating cells from patient tumor samples. Therapeutic synergies, when normal astrocytes were used as a toxicity cell model, reinforced the pharmacological interest regarding their potential clinical use. CONCLUSIONS: COMBImage enables, for the first time, fast and optimized pairwise drug combination analyses of temporal changes in label-free video microscopy movies. Providing this jointly with conventional cell viability based end point analyses, it could help accelerating and guiding any drug discovery and development project, without use of cell labeling and the need to employ a particular live cell imaging instrument.

Targeting tumor cells based on Phosphodiesterase 3A expression.

We and others have previously reported a correlation between high phosphodiesterase 3A (PDE3A) expression and selective sensitivity to phosphodiesterase (PDE) inhibitors. This indicates that PDE3A could serve both as a drug target and a biomarker of sensitivity to PDE3 inhibition. In this report, we explored publicly available mRNA gene expression data to identify cell lines with different PDE3A expression. Cell lines with high PDE3A expression showed marked in vitro sensitivity to PDE inhibitors zardaverine and quazinone, when compared with those having low PDE3A expression. Immunofluorescence and immunohistochemical stainings were in agreement with PDE3A mRNA expression, providing suitable alternatives for biomarker analysis of clinical tissue specimens. Moreover, we here demonstrate that tumor cells from patients with ovarian carcinoma show great variability in PDE3A protein expression and that level of PDE3A expression is correlated with sensitivity to PDE inhibition. Finally, we demonstrate that PDE3A is highly expressed in subsets of patient tumor cell samples from different solid cancer diagnoses and expressed at exceptional levels in gastrointestinal stromal tumor (GIST) specimens. Importantly, vulnerability to PDE3 inhibitors has recently been associated with co-expression of PDE3A and Schlafen family member 12 (SLFN12). We here demonstrate that high expression of PDE3A in clinical specimens, at least on the mRNA level, seems to be frequently associated with high SLFN12 expression. In conclusion, PDE3A seems to be both a promising biomarker and drug target for individualized drug treatment of various cancers.

Fast multicolor 3D imaging using aberration-corrected multifocus microscopy.

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.

Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination.

Previous implementations of structured-illumination microscopy (SIM) were slow or designed for one-color excitation, sacrificing two unique and extremely beneficial aspects of light microscopy: live-cell imaging in multiple colors. This is especially unfortunate because, among the resolution-extending techniques, SIM is an attractive choice for live-cell imaging; it requires no special fluorophores or high light intensities to achieve twice diffraction-limited resolution in three dimensions. Furthermore, its wide-field nature makes it light-efficient and decouples the acquisition speed from the size of the lateral field of view, meaning that high frame rates over large volumes are possible. Here, we report a previously undescribed SIM setup that is fast enough to record 3D two-color datasets of living whole cells. Using rapidly programmable liquid crystal devices and a flexible 2D grid pattern algorithm to switch between excitation wavelengths quickly, we show volume rates as high as 4 s in one color and 8.5 s in two colors over tens of time points. To demonstrate the capabilities of our microscope, we image a variety of biological structures, including mitochondria, clathrin-coated vesicles, and the actin cytoskeleton, in either HeLa cells or cultured neurons.

Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution.

Using ultralow light intensities that are well suited for investigating biological samples, we demonstrate whole-cell superresolution imaging by nonlinear structured-illumination microscopy. Structured-illumination microscopy can increase the spatial resolution of a wide-field light microscope by a factor of two, with greater resolution extension possible if the emission rate of the sample responds nonlinearly to the illumination intensity. Saturating the fluorophore excited state is one such nonlinear response, and a realization of this idea, saturated structured-illumination microscopy, has achieved approximately 50-nm resolution on dye-filled polystyrene beads. Unfortunately, because saturation requires extremely high light intensities that are likely to accelerate photobleaching and damage even fixed tissue, this implementation is of limited use for studying biological samples. Here, reversible photoswitching of a fluorescent protein provides the required nonlinearity at light intensities six orders of magnitude lower than those needed for saturation. We experimentally demonstrate approximately 40-nm resolution on purified microtubules labeled with the fluorescent photoswitchable protein Dronpa, and we visualize cellular structures by imaging the mammalian nuclear pore and actin cytoskeleton. As a result, nonlinear structured-illumination microscopy is now a biologically compatible superresolution imaging method.

Label free high throughput screening for apoptosis inducing chemicals using time-lapse microscopy signal processing.

Label free time-lapse microscopy has opened a new avenue to the study of time evolving events in living cells. When combined with automated image analysis it provides a powerful tool that enables automated large-scale spatiotemporal quantification at the cell population level. Very few attempts, however, have been reported regarding the design of image analysis algorithms dedicated to the detection of apoptotic cells in such time-lapse microscopy images. In particular, none of the reported attempts is based on sufficiently fast signal processing algorithms to enable large-scale detection of apoptosis within hours/days without access to high-end computers. Here we show that it is indeed possible to successfully detect chemically induced apoptosis by applying a two-dimensional linear matched filter tailored to the detection of objects with the typical features of an apoptotic cell in phase-contrast images. First a set of recorded computational detections of apoptosis was validated by comparison with apoptosis specific caspase activity readouts obtained via a fluorescence based assay. Then a large screen encompassing 2,866 drug like compounds was performed using the human colorectal carcinoma cell line HCT116. In addition to many well known inducers (positive controls) the screening resulted in the detection of two compounds here reported for the first time to induce apoptosis.

Super-resolution 3D microscopy of live whole cells using structured illumination.

Three-dimensional (3D) structured-illumination microscopy (SIM) can double the lateral and axial resolution of a wide-field fluorescence microscope but has been too slow for live imaging. Here we apply 3D SIM to living samples and record whole cells at up to 5 s per volume for >50 time points with 120-nm lateral and 360-nm axial resolution. We demonstrate the technique by imaging microtubules in S2 cells and mitochondria in HeLa cells.

Automated QuantMap for rapid quantitative molecular network topology analysis.

SUMMARY: The previously disclosed QuantMap method for grouping chemicals by biological activity used online services for much of the data gathering and some of the numerical analysis. The present work attempts to streamline this process by using local copies of the databases and in-house analysis. Using computational methods similar or identical to those used in the previous work, a qualitatively equivalent result was found in just a few seconds on the same dataset (collection of 18 drugs). We use the user-friendly Galaxy framework to enable users to analyze their own datasets. Hopefully, this will make the QuantMap method more practical and accessible and help achieve its goals to provide substantial assistance to drug repositioning, pharmacology evaluation and toxicology risk assessment. AVAILABILITY: http://galaxy.predpharmtox.org CONTACT: mats.gustafsson@medsci.uu.se or ola.spjuth@farmbio.uu.se SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

NMR spectroscopy-based metabolic profiling of drug-induced changes in vitro can discriminate between pharmacological classes.

Drug-induced changes in mammalian cell line models have already been extensively profiled at the systemic mRNA level and subsequently used to suggest mechanisms of action for new substances, as well as to support drug repurposing, i.e., identifying new potential indications for drugs already licensed for other pharmacotherapy settings. The seminal work in this field, which includes a large database and computational algorithms for pattern matching, is known as the "Connectivity Map" (CMap). However, the potential of similar exercises at the metabolite level is still largely unexplored. Only recently, the first high-throughput metabolomic assay pilot study was published, which involved screening the metabolic response to a set of 56 kinase inhibitors in a 96-well format. Here, we report results from a separately developed metabolic profiling assay, which leverages (1)H NMR spectroscopy to the quantification of metabolic changes in the HCT116 colorectal cancer cell line, in response to each of 26 compounds. These agents are distributed across 12 different pharmacological classes covering a broad spectrum of bioactivity. Differential metabolic profiles, inferred from multivariate spectral analysis of 18 spectral bins, allowed clustering of the most-tested drugs, according to their respective pharmacological class. A more-advanced supervised analysis, involving one multivariate scattering matrix per pharmacological class and using only 3 spectral bins (3 metabolites), showed even more distinct pharmacology-related cluster formations. In conclusion, this type of relatively fast and inexpensive profiling seems to provide a promising alternative to that afforded by mRNA expression analysis, which is relatively slow and costly. As also indicated by the present pilot study, the resulting metabolic profiles do not seem to provide as information-rich signatures as those obtained using systemic mRNA profiling, but the methodology holds strong promise for significant refinement.

Comprehensive approach to national tire wear emissions: Challenges and implications.

The use of vehicle tires has been identified as a major source of microplastics in the environment and an increasing source of urban particulate air pollution. In light of increasing traffic volumes, increasingly heavier and more powerful vehicles due to trends and electrification, and the lack of tire wear regulation, methods to estimate and monitor changes in national emissions are needed as input for environmental impact assessments. Emission estimations of tire wear are made either based on the mileage approach or the sales approach. This study aims to investigate if and how the mileage approach can be improved by using emission factors for passenger cars and LDVs based on our own measurements and emission factors from the literature for HDVs and buses. An approach with emission factor adjustments based on weight and number of tires in combination with highly detailed mileage data has been evaluated. Sales approach calculations have been used to validate the method. A secondary aim was to use the new mileage approach framework to calculate the national tire wear emissions for Sweden. These calculations resulted in slightly lower total emissions than previous estimations provide, but with higher emissions for passenger cars and light-duty vehicles, and lower emissions for heavy-duty vehicles and motorcycles. Passenger cars constitute more than half of the total emissions. It is concluded that even though the framework offers greater detail, thus increasing the possibilities to adjust for changes in emission factors and mileages in specific vehicle categories, the challenges posed by such factors as the lack of measured emission factors for heavy-duty vehicles and uncertainties regarding the quality of mileage statistics makes the estimations uncertain. Important future suggestions for research include establishing reliable emission factors, especially for heavy-duty vehicles, and initiating research to better understand how climate, road networks, surface properties, and vehicle fleet characteristics affect emission factors.

Exhaustive in vitro evaluation of the 9-drug cocktail CUSP9 for treatment of glioblastoma.

The CUSP9 protocol is a polypharmaceutical strategy aiming at addressing the complexity of glioblastoma by targeting multiple pathways. Although the rationale for this 9-drug cocktail is well-supported by theoretical and in vitro data, its effectiveness compared to its 511 possible subsets has not been comprehensively evaluated. Such an analysis could reveal if fewer drugs could achieve similar or better outcomes. We conducted an exhaustive in vitro evaluation of the CUSP9 protocol using COMBImageDL, our specialized framework for testing higher-order drug combinations. This study assessed all 511 subsets of the CUSP9v3 protocol, in combination with temozolomide, on two clonal cultures of glioma-initiating cells derived from patient samples. The drugs were used at fixed, clinically relevant concentrations, and the experiment was performed in quadruplicate with endpoint cell viability and live-cell imaging readouts. Our results showed that several lower-order drug combinations produced effects equivalent to the full CUSP9 cocktail, indicating potential for simplified regimens in personalized therapy. Further validation through in vivo and precision medicine testing is required. Notably, a subset of four drugs (auranofin, disulfiram, itraconazole, sertraline) was particularly effective, reducing cell growth, altering cell morphology, increasing apoptotic-like cells within 4-28 h, and significantly decreasing cell viability after 68 h compared to untreated cells. This study underscores the importance and feasibility of comprehensive in vitro evaluations of complex drug combinations on patient-derived tumor cells, serving as a critical step toward (pre-)clinical development.

Screening for phenotype selective activity in multidrug resistant cells identifies a novel tubulin active agent insensitive to common forms of cancer drug resistance.

BACKGROUND: Drug resistance is a common cause of treatment failure in cancer patients and encompasses a multitude of different mechanisms. The aim of the present study was to identify drugs effective on multidrug resistant cells. METHODS: The RPMI 8226 myeloma cell line and its multidrug resistant subline 8226/Dox40 was screened for cytotoxicity in response to 3,000 chemically diverse compounds using a fluorometric cytotoxicity assay (FMCA). Follow-up profiling was subsequently performed using various cellular and biochemical assays. RESULTS: One compound, designated VLX40, demonstrated a higher activity against 8226/Dox40 cells compared to its parental counterpart. VLX40 induced delayed cell death with apoptotic features. Mechanistic exploration was performed using gene expression analysis of drug exposed tumor cells to generate a drug-specific signature. Strong connections to tubulin inhibitors and microtubule cytoskeleton were retrieved. The mechanistic hypothesis of VLX40 acting as a tubulin inhibitor was confirmed by direct measurements of interaction with tubulin polymerization using a biochemical assay and supported by demonstration of G2/M cell cycle arrest. When tested against a broad panel of primary cultures of patient tumor cells (PCPTC) representing different forms of leukemia and solid tumors, VLX40 displayed high activity against both myeloid and lymphoid leukemias in contrast to the reference compound vincristine to which myeloid blast cells are often insensitive. Significant in vivo activity was confirmed in myeloid U-937 cells implanted subcutaneously in mice using the hollow fiber model. CONCLUSIONS: The results indicate that VLX40 may be a useful prototype for development of novel tubulin active agents that are insensitive to common mechanisms of cancer drug resistance.

Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material.

A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pm sensitivity each consuming only 1 µl of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.