OBJECTIVE: The highly infiltrative growth of glioblastoma (GBM) makes distinction between the tumor and normal brain tissue challenging. Therefore, fluorescence-guided surgery is often used to improve visual identification of radiological tumor margins. The aim of this study was to evaluate the ability of recently developed molecularly targeted near-infrared (NIR) protease-activated probes to visualize GBM tissue and to compare the most promising candidate with the gold standard, 5-aminolevulinic acid (5-ALA). METHODS: Single-substrate probes 6QC-ICG and 6QC-Cy5 (cysteine cathepsin cleavable), double-substrate probes AG2-FNIR and AG2-Cy5 (cysteine cathepsin and caspase 3 cleavable), and 5-ALA were administered intravenously to mice with orthotopic tumors. Activation of the probes was also evaluated in cell cultures in vitro and in biopsy material from patients with GBM ex vivo. The tumor to normal brain tissue fluorescence ratio (TNR) was quantified in brain sections using preclinical and clinical visualization platforms, and in tissue homogenates and cell suspensions using spectrofluorimetry. Subcellular localization of the fluorophores was visualized by confocal microscopy. RESULTS: In vitro, the single-substrate probe 6QC-ICG was cleaved in glioma cells and macrophages, and the resulting fluorophore accumulated intracellularly. In experimental GBMs, both single- and double-substrate probes visualized tumor tissue, while in healthy brain tissue the signal was minimal. TNR was highest for 6QC-ICG and AG2-FNIR, but the signal intensity was higher for 6QC-ICG. Using xenograft and syngeneic mouse models, as well as human GBM biopsy material ex vivo, the authors confirmed the ability of 6QC-ICG to specifically visualize the glioma tissue using preclinical and clinical visualization platforms. Finally, a comparison with 5-ALA in animals coadministered with both compounds revealed a higher TNR for 6QC-ICG in experimental GBMs. CONCLUSIONS: The cysteine cathepsin-cleavable probe 6QC-ICG is activated by glioma cells and tumor-associated macrophages, leading to a high contrast between tumor and nontumorous brain tissue that is superior to that of the current standard, 5-ALA. In addition to a well-defined mechanism of action, protease-activated probes that use NIR fluorophores (e.g., indocyanine green) have the advantage of low absorption and scattering of the NIR light and lower tissue autofluorescence. These results suggest that 6QC-ICG has the potential to become the targeted agent in intraoperative detection of GBM tissue using fluorescence imaging.
- MeSH
- fluorescenční barviva MeSH
- glioblastom * diagnostické zobrazování patologie MeSH
- kyselina aminolevulová * MeSH
- lidé MeSH
- molekulární sondy MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory mozku * diagnostické zobrazování patologie MeSH
- optické zobrazování metody MeSH
- proteasy metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with μ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.
- MeSH
- adaptorový proteinový komplex - alfa-podjednotky genetika MeSH
- adaptorový proteinový komplex 2 genetika metabolismus MeSH
- endocytóza genetika MeSH
- fosforylace genetika MeSH
- klathrin genetika metabolismus MeSH
- klathrinové vezikuly genetika metabolismus MeSH
- lidé MeSH
- potažené jamky v buněčné membráně genetika metabolismus MeSH
- třídící nexiny genetika MeSH
- vazba proteinů genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PURPOSE: Breast ultrasonography (US) presents an alternative to mammography in young asymptomatic individuals and a complementary examination in screening of women with dense breasts. Handheld US is the standard-of-care, yet when used in whole-breast examination, no effort has been devoted to monitoring breast coverage and missed regions, which is the purpose of this study. METHODS: We introduce a computer-aided system assisting radiologists and US technologists in covering the whole breast with minimum alteration to the standard workflow. The proposed system comprises a standard US device, proprietary electromagnetic 3D tracking technology and software that combines US visual and tracking data to estimate a probe trajectory, total time spent in different breast segments, and a map of missed regions. A case study, which involved four radiologists (two junior and two senior) performing whole-breast ultrasound in 75 asymptomatic patients, was conducted to test the importance and relevance of the system. RESULTS: The mean process time per breast was [Formula: see text], with no statistically significant difference between the left and the right sides, and slightly longer examination time of junior radiologists. The process time density shows that central parts of the breast have better coverage compared to the periphery. Within the central part, missed regions of minimum detectable size of [Formula: see text] occur in [Formula: see text] of examinations, and non-negligible [Formula: see text] regions occur in [Formula: see text] of cases. CONCLUSION: The results of the case study indicate that missed regions are present in handheld whole-breast US, which renders the proposed system for tracking the probe position during examination a valuable tool for monitoring coverage.
- MeSH
- design vybavení MeSH
- diagnóza počítačová * MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mamografie metody MeSH
- nádory prsu diagnostické zobrazování MeSH
- počítače do ruky MeSH
- počítačové systémy MeSH
- počítačové zpracování obrazu MeSH
- prsy diagnostické zobrazování MeSH
- reprodukovatelnost výsledků MeSH
- software MeSH
- ultrasonografie prsů metody MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Biocompatibility testing of new materials is often performed in vitro by measuring the growth rate of mammalian cancer cells in time-lapse images acquired by phase contrast microscopes. The growth rate is measured by tracking cell coverage, which requires an accurate automatic segmentation method. However, cancer cells have irregular shapes that change over time, the mottled background pattern is partially visible through the cells and the images contain artifacts such as halos. We developed a novel algorithm for cell segmentation that copes with the mentioned challenges. It is based on temporal differences of consecutive images and a combination of thresholding, blurring, and morphological operations. We tested the algorithm on images of four cell types acquired by two different microscopes, evaluated the precision of segmentation against manual segmentation performed by a human operator, and finally provided comparison with other freely available methods. We propose a new, fully automated method for measuring the cell growth rate based on fitting a coverage curve with the Verhulst population model. The algorithm is fast and shows accuracy comparable with manual segmentation. Most notably it can correctly separate live from dead cells.
Retinal images are essential clinical resources for the diagnosis of retinopathy and many other ocular diseases. Because of improper acquisition conditions or inherent optical aberrations in the eye, the images are often degraded with blur. In many common cases, the blur varies across the field of view. Most image deblurring algorithms assume a space-invariant blur, which fails in the presence of space-variant (SV) blur. In this work, we propose an innovative strategy for the restoration of retinal images in which we consider the blur to be both unknown and SV. We model the blur by a linear operation interpreted as a convolution with a point-spread function (PSF) that changes with the position in the image. To achieve an artifact-free restoration, we propose a framework for a robust estimation of the SV PSF based on an eye-domain knowledge strategy. The restoration method was tested on artificially and naturally degraded retinal images. The results show an important enhancement, significant enough to leverage the images' clinical use.
- MeSH
- algoritmy MeSH
- angiografie metody MeSH
- artefakty MeSH
- astigmatismus diagnóza MeSH
- diagnostické techniky oftalmologické * MeSH
- fundus oculi MeSH
- lidé MeSH
- normální rozdělení MeSH
- optika a fotonika MeSH
- počítačové zpracování obrazu MeSH
- reprodukovatelnost výsledků MeSH
- retina patologie MeSH
- retinální cévy patologie MeSH
- rozpoznávání automatizované metody MeSH
- statistické modely MeSH
- zrak MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Blind deconvolution, which comprises simultaneous blur and image estimations, is a strongly ill-posed problem. It is by now well known that if multiple images of the same scene are acquired, this multichannel (MC) blind deconvolution problem is better posed and allows blur estimation directly from the degraded images. We improve the MC idea by adding robustness to noise and stability in the case of large blurs or if the blur size is vastly overestimated. We formulate blind deconvolution as an l(1) -regularized optimization problem and seek a solution by alternately optimizing with respect to the image and with respect to blurs. Each optimization step is converted to a constrained problem by variable splitting and then is addressed with an augmented Lagrangian method, which permits simple and fast implementation in the Fourier domain. The rapid convergence of the proposed method is illustrated on synthetically blurred data. Applicability is also demonstrated on the deconvolution of real photos taken by a digital camera.
- MeSH
- algoritmy MeSH
- artefakty MeSH
- interpretace obrazu počítačem metody MeSH
- reprodukovatelnost výsledků MeSH
- rozpoznávání automatizované metody MeSH
- senzitivita a specificita MeSH
- vylepšení obrazu metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
