The use of optical coherence tomography (OCT) to study ocular diseases associated with choroidal physiology is sharply limited by the lack of available automated segmentation tools. Current research ...largely relies on hand-traced, single B-Scan segmentations because commercially available programs require high quality images, and the existing implementations are closed, scarce and not freely available. We developed and implemented a robust algorithm for segmenting and quantifying the choroidal layer from 3-dimensional OCT reconstructions. Here, we describe the algorithm, validate and benchmark the results, and provide an open-source implementation under the General Public License for any researcher to use (https://www.mathworks.com/matlabcentral/fileexchange/61275-choroidsegmentation).
We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) that relies on complete analysis of both the temporal and spatial correlation lags ...for intensity fluctuations from a laser-scanning microscopy image series. This new approach allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) for fluorescently labeled membrane proteins in living cells through monitoring of the time evolution of the full space-time correlation function. By using filtering in Fourier space to remove frequencies associated with immobile components, we are able to measure the protein transport even in the presence of a large fraction (>90%) of immobile species. We present the background theory, computer simulations, and analysis of measurements on fluorescent microspheres to demonstrate proof of principle, capabilities, and limitations of the method. We demonstrate mapping of flow vectors for mixed samples containing fluorescent microspheres with different emission wavelengths using space time image cross-correlation. We also present results from two-photon laser-scanning microscopy studies of
α-actinin/enhanced green fluorescent protein fusion constructs at the basal membrane of living CHO cells. Using space-time image correlation spectroscopy (STICS), we are able to measure protein fluxes with magnitudes of
μm/min from retracting lamellar regions and protrusions for adherent cells. We also demonstrate the measurement of correlated directed flows (magnitudes of
μm/min) and diffusion of interacting
α5 integrin/enhanced cyan fluorescent protein and
α-actinin/enhanced yellow fluorescent protein within living CHO cells. The STICS method permits us to generate complete transport maps of proteins within subregions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements.
The identification of eye diseases and their progression often relies on a clear visualization of the anatomy and on different metrics extracted from Optical Coherence Tomography (OCT) B-scans. ...However, speckle noise hinders the quality of rapid OCT imaging, hampering the extraction and reliability of biomarkers that require time series. By synchronizing the acquisition of OCT images with the timing of the cardiac pulse, we transform a low-quality OCT video into a clear version by phase-wrapping each frame to the heart pulsation and averaging frames that correspond to the same instant in the cardiac cycle. Here, we compare the performance of our one-cycle denoising strategy with a deep-learning architecture, Noise2Noise, as well as classical denoising methods such as BM3D and Non-Local Means (NLM). We systematically analyze different image quality descriptors as well as region-specific metrics to assess the denoising performance based on the anatomy of the eye. The one-cycle method achieves the highest denoising performance, increases image quality and preserves the high-resolution structures within the eye tissues. The proposed workflow can be readily implemented in a clinical setting.
Mechanisms regulating B cell development, activation, education in the germinal center (GC) and differentiation, underpin the humoral immune response. Protein arginine methyltransferase 5 (Prmt5), ...which catalyzes most symmetric dimethyl arginine protein modifications, is overexpressed in B cell lymphomas but its function in normal B cells is poorly defined. Here we show that Prmt5 is necessary for antibody responses and has essential but distinct functions in all proliferative B cell stages in mice. Prmt5 is necessary for B cell development by preventing p53-dependent and p53-independent blocks in Pro-B and Pre-B cells, respectively. By contrast, Prmt5 protects, via p53-independent pathways, mature B cells from apoptosis during activation, promotes GC expansion, and counters plasma cell differentiation. Phenotypic and RNA-seq data indicate that Prmt5 regulates GC light zone B cell fate by regulating transcriptional programs, achieved in part by ensuring RNA splicing fidelity. Our results establish Prmt5 as an essential regulator of B cell biology.
Intrinsic and acquired resistance to cisplatin remains a primary hurdle to treatment of high-grade serous ovarian cancer (HGSOC). Cisplatin selectively kills tumor cells by inducing DNA crosslinks ...that block replicative DNA polymerases. Single-stranded DNA (ssDNA) generated at resulting stalled replication forks (RF) is bound and protected by heterotrimeric replication protein A (RPA), which then serves as a platform for recruitment and activation of replication stress response factors. Cells deficient in this response are characterized by extensive ssDNA formation and excessive RPA recruitment that exhausts the available pool of RPA, which (i) inhibits RPA-dependent processes such as nucleotide excision repair (NER) and (ii) causes catastrophic failure of blocked RF. Here, we investigated the influence of RPA availability on chemosensitivity using a panel of human HGSOC cell lines. Our data revealed a striking correlation among these cell lines between cisplatin sensitivity and the inability to efficiently repair DNA via NER, specifically during S phase. Such defects in NER were attributable to RPA exhaustion arising from aberrant activation of DNA replication origins during replication stress. Reduced RPA availability promoted Mre11-dependent degradation of nascent DNA at stalled RF in cell lines exhibiting elevated sensitivity to cisplatin. Strikingly, defective S-phase NER, RF instability, and cisplatin sensitivity could all be rescued by ectopic overexpression of RPA. Taken together, our findings indicate that RPA exhaustion represents a major determinant of cisplatin sensitivity in HGSOC cell lines.
The influence of replication protein A exhaustion on cisplatin sensitivity harbors important implications toward improving therapy of various cancers that initially respond to platinum-based agents but later relapse due to intrinsic or acquired drug resistance.
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Ocular rigidity (OR) is thought to play a role in the pathogenesis of glaucoma, but the lack of reliable non-invasive measurements has been a major technical challenge. We recently developed a ...clinical method using optical coherence tomography time-lapse imaging and automated choroidal segmentation to measure the pulsatile choroidal volume change (ΔV) and calculate OR using Friedenwald's equation. Here we assess the validity and repeatability of this non-invasive technique. We also propose an improved mathematical model of choroidal thickness to extrapolate ΔV from the pulsatile submacular choroidal thickness change more accurately. The new mathematical model uses anatomical data accounting for the choroid thickness near the equator. The validity of the technique was tested by comparing OR coefficients obtained using our non-invasive method (OROCT) and those obtained with an invasive procedure involving intravitreal injections of Bevacizumab (ORIVI) in 12 eyes. Intrasession and intersession repeatability was assessed for 72 and 8 eyes respectively with two consecutive measurements of OR. Using the new mathematical model, we obtained OR values which are closer to those obtained using the invasive procedure and previously reported techniques. A regression line was calculated to predict the ORIVI based on OROCT, such that ORIVI = 0.655 × OROCT. A strong correlation between OROCT and ORIVI was found, with a Spearman coefficient of 0.853 (p < 0.001). The intraclass correlation coefficient for intrasession and intersession repeatability was 0.925, 95% CI 0.881, 0.953 and 0.950, 95% CI 0.763, 0.990 respectively. This confirms the validity and good repeatability of OR measurements using our non-invasive clinical method.
•Clinical validation of a non-invasive method for measuring ocular rigidity in vivo.•Strong correlation between OR coefficients obtained non-invasively and invasively.•Integrated choroidal anatomy to improve our mathematical model and OR measurements.•The method is reproducible and can be used to measure OR accurately.
The performance of the single particle tracking (SPT) nearest-neighbor algorithm is determined by parameters that need to be set according to the characteristics of the time series under study. ...Inhomogeneous systems, where these characteristics fluctuate spatially, are poorly tracked when parameters are set globally.
We present a novel SPT approach that adapts the well-known nearest-neighbor tracking algorithm to the local density of particles to overcome the problems of inhomogeneity.
We demonstrate the performance improvement provided by the proposed method using numerical simulations and experimental data and compare its performance with state of the art SPT algorithms.
The algorithms proposed here, are released under the GNU General Public License and are freely available on the web at http://sourceforge.net/p/adaptivespt.
javier.mazzaferri@gmail.com
Supplementary data are available at Bioinformatics online.
Neutrophil recruitment guided by chemotactic cues is a central event in host defense against infection and tissue injury. While the mechanisms underlying neutrophil chemotaxis have been extensively ...studied, these are just recently being addressed by using high-content approaches or surface-bound chemotactic gradients (haptotaxis) in vitro. Here, we report a haptotaxis assay, based on the classic under-agarose assay, which combines an optical patterning technique to generate surface-bound formyl peptide gradients as well as an automated imaging and analysis of a large number of migration trajectories. We show that human neutrophils migrate on covalently-bound formyl-peptide gradients, which influence the speed and frequency of neutrophil penetration under the agarose. Analysis revealed that neutrophils migrating on surface-bound patterns accumulate in the region of the highest peptide concentration, thereby mimicking in vivo events. We propose the use of a chemotactic precision index, gyration tensors and neutrophil penetration rate for characterizing haptotaxis. This high-content assay provides a simple approach that can be applied for studying molecular mechanisms underlying haptotaxis on user-defined gradient shape.
Promoting axon regeneration following injury is one of the ultimate challenges of neuroscience, and understanding the mechanisms that regulate axon growth and guidance is essential to achieve this ...goal. During development axons are directed over relatively long distances by a precise extracellular distribution of chemical signals in the embryonic nervous system. Multiple guidance proteins, including netrins, slits, semaphorins, ephrins and neurotrophins have been identified as key players in this process. During the last decade, engineered cell culture substrates have been developed to investigate the cellular and molecular mechanisms underlying axon guidance. This review is focused on the biological insights that have been achieved using new techniques that attempt to mimic
in vitro
the spatial patterns of proteins that growth cones encounter
in vivo
.
During the last decade, engineered cell culture substrates have been developed to investigate the cellular and molecular mechanisms underlying axon guidance. This review is focused on the biological insights that have been achieved using new techniques that attempt to mimic
in vitro
the spatial patterns of proteins that growth cones encounter
in vivo
.
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