IsletSwipe, a mobile platform for expert opinion exchange on islet graft images.

We previously developed a deep learning-based web service (IsletNet) for an automated counting of isolated pancreatic islets. The neural network training is limited by the absent consensus on the ground truth annotations. Here, we present a platform (IsletSwipe) for an exchange of graphical opinions among experts to facilitate the consensus formation. The platform consists of a web interface and a mobile application. In a small pilot study, we demonstrate the functionalities and the use case scenarios of the platform. Nine experts from three centers validated the drawing tools, tested precision and consistency of the expert contour drawing, and evaluated user experience. Eight experts from two centers proceeded to evaluate additional images to demonstrate the following two use case scenarios. The Validation scenario involves an automated selection of images and islets for the expert scrutiny. It is scalable (more experts, images, and islets may readily be added) and can be applied to independent validation of islet contours from various sources. The Inquiry scenario serves the ground truth generating expert in seeking assistance from peers to achieve consensus on challenging cases during the preparation for IsletNet training. This scenario is limited to a small number of manually selected images and islets. The experts gained an opportunity to influence IsletNet training and to compare other experts' opinions with their own. The ground truth-generating expert obtained feedback for future IsletNet training. IsletSwipe is a suitable tool for the consensus finding. Experts from additional centers are welcome to participate.

Nicotinic Acetylcholine Receptors Expressed by Striatal Interneurons Inhibit Striatal Activity and Control Striatal-Dependent Behaviors.

Acetylcholine is an important modulator of striatal activity, and it is vital to controlling striatal-dependent behaviors, including motor and cognitive functions. Despite this significance, the mechanisms determining how acetylcholine impacts striatal signaling are still not fully understood. In particular, little is known about the role of nAChRs expressed by striatal interneurons. In the present study, we used FISH to determine which neuronal types express the most prevalent beta2 nicotinic subunit in the mouse striatum. Our data support a common view that nAChR expression is mostly restricted to striatal interneurons. Surprisingly though, cholinergic interneurons were identified as a population with the highest expression of beta2 nicotinic subunit. To investigate the functional significance of beta2-containing nAChRs in striatal interneurons, we deleted them by injecting the AAV-Cre vector into the striatum of beta2-flox/flox male mice. The deletion led to alterations in several behavioral domains, namely, to an increased anxiety-like behavior, decrease in sociability ratio, deficit in discrimination learning, and increased amphetamine-induced hyperlocomotion and c-Fos expression in mice with beta2 deletion. Further colocalization analysis showed that the increased c-Fos expression was present in both medium spiny neurons and presumed striatal interneurons. The present study concludes that, despite being relatively rare, beta2-containing nAChRs are primarily expressed in striatal neurons by cholinergic interneurons and play a significant role in behavior.SIGNIFICANCE STATEMENT A large variety of nAChRs are expressed in the striatum, a brain region that is crucial in the control of behavior. The complexity of receptors with different functions is hindering our understanding of mechanisms through which striatal acetylcholine modulates behavior. We focused on the role of a small population of beta2-containing nAChRs. We identified neuronal types expressing these receptors and determined their impact in the control of explorative behavior, anxiety-like behavior, learning, and sensitivity to stimulants. Additional experiments showed that these alterations were associated with an overall increased activity of striatal neurons. Thus, the small population of nicotinic receptors represents an interesting target for a modulation of response to stimulant drugs and other striatal-based behavior.

Wnt/ß-Catenin Signaling Promotes Differentiation of Ischemia-Activated Adult Neural Stem/Progenitor Cells to Neuronal Precursors.

Modulating endogenous regenerative processes may represent a suitable treatment for central nervous system (CNS) injuries, such as stroke or trauma. Neural stem/progenitor cells (NS/PCs), which naturally reside in the subventricular zone (SVZ) of the adult brain, proliferate and differentiate to other cell types, and therefore may compensate the negative consequences of ischemic injury. The fate of NS/PCs in the developing brain is largely influenced by Wingless/Integrated (Wnt) signaling; however, its role in the differentiation of adult NS/PCs under ischemic conditions is still enigmatic. In our previous study, we identified the Wnt/ß-catenin signaling pathway as a factor promoting neurogenesis at the expense of gliogenesis in neonatal mice. In this study, we used adult transgenic mice in order to assess the impact of the canonical Wnt pathway modulation (inhibition or hyper-activation) on NS/PCs derived from the SVZ, and combined it with the middle cerebral artery occlusion (MCAO) to disclose the effect of focal cerebral ischemia (FCI). Based on the electrophysiological properties of cultured cells, we first identified three cell types that represented in vitro differentiated NS/PCs - astrocytes, neuron-like cells, and precursor cells. Following FCI, we detected fewer neuron-like cells after Wnt signaling inhibition. Furthermore, the immunohistochemical analysis revealed an overall higher expression of cell-type-specific proteins after FCI, indicating increased proliferation and differentiation rates of NS/PCs in the SVZ. Remarkably, Wnt signaling hyper-activation increased the abundance of proliferating and neuron-like cells, while Wnt pathway inhibition had the opposite effect. Finally, the expression profiling at the single cell level revealed an increased proportion of neural stem cells and neuroblasts after FCI. These observations indicate that Wnt signaling enhances NS/PCs-based regeneration in the adult mouse brain following FCI, and supports neuronal differentiation in the SVZ.

Regulation of Microtubule Nucleation in Mouse Bone Marrow-Derived Mast Cells by Protein Tyrosine Phosphatase SHP-1.

The antigen-mediated activation of mast cells initiates signaling events leading to their degranulation, to the release of inflammatory mediators, and to the synthesis of cytokines and chemokines. Although rapid and transient microtubule reorganization during activation has been described, the molecular mechanisms that control their rearrangement are largely unknown. Microtubule nucleation is mediated by γ-tubulin complexes. In this study, we report on the regulation of microtubule nucleation in bone marrow-derived mast cells (BMMCs) by Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1; Ptpn6). Reciprocal immunoprecipitation experiments and pull-down assays revealed that SHP-1 is present in complexes containing γ-tubulin complex proteins and protein tyrosine kinase Syk. Microtubule regrowth experiments in cells with deleted SHP-1 showed a stimulation of microtubule nucleation, and phenotypic rescue experiments confirmed that SHP-1 represents a negative regulator of microtubule nucleation in BMMCs. Moreover, the inhibition of the SHP-1 activity by inhibitors TPI-1 and NSC87877 also augmented microtubule nucleation. The regulation was due to changes in γ-tubulin accumulation. Further experiments with antigen-activated cells showed that the deletion of SHP-1 stimulated the generation of microtubule protrusions, the activity of Syk kinase, and degranulation. Our data suggest a novel mechanism for the suppression of microtubule formation in the later stages of mast cell activation.

Nuclear pore protein TPR associates with lamin B1 and affects nuclear lamina organization and nuclear pore distribution.

The organization of the nuclear periphery is crucial for many nuclear functions. Nuclear lamins form dense network at the nuclear periphery and play a substantial role in chromatin organization, transcription regulation and in organization of nuclear pore complexes (NPCs). Here, we show that TPR, the protein located preferentially within the nuclear baskets of NPCs, associates with lamin B1. The depletion of TPR affects the organization of lamin B1 but not lamin A/C within the nuclear lamina as shown by stimulated emission depletion microscopy. Finally, reduction of TPR affects the distribution of NPCs within the nuclear envelope and the effect can be reversed by simultaneous knock-down of lamin A/C or the overexpression of lamin B1. Our work suggests a novel role for the TPR at the nuclear periphery: the TPR contributes to the organization of the nuclear lamina and in cooperation with lamins guards the interphase assembly of nuclear pore complexes.

Nuclear phosphatidylinositol 4,5-bisphosphate islets contribute to efficient RNA polymerase II-dependent transcription.

This paper describes a novel type of nuclear structure - nuclear lipid islets (NLIs). They are of 40-100 nm with a lipidic interior, and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] molecules comprise a significant part of their surface. Most of NLIs have RNA at the periphery. Consistent with that, RNA is required for their integrity. The NLI periphery is associated with Pol II transcription machinery, including the largest Pol II subunit, transcription factors and NM1 (also known as NMI). The PtdIns(4,5)P2-NM1 interaction is important for Pol II transcription, since NM1 knockdown reduces the Pol II transcription level, and the overexpression of wild-type NM1 [but not NM1 mutated in the PtdIns(4,5)P2-binding site] rescues the transcription. Importantly, Pol II transcription is dependent on NLI integrity, because an enzymatic reduction of the PtdIns(4,5)P2 level results in a decrease of the Pol II transcription level. Furthermore, about half of nascent transcripts localise to NLIs, and transcriptionally active transgene loci preferentially colocalise with NLIs. We hypothesize that NLIs serve as a structural platform that facilitates the formation of Pol II transcription factories, thus participating in the formation of nuclear architecture competent for transcription.

3D reconstruction of peripheral nerves from optical projection tomography images: A method for studying fascicular interconnections and intraneural plexuses.

The general microscopic characteristics of nerves are described in several textbooks of histology, but the specific microanatomies of most nerves that can be blocked by anesthesiologists are usually less well known. Our objective was to evaluate the 3D reconstruction of nerve fascicles from optical projection tomography images (OPT) and the ability to undertake an internal navigation exploring the morphology in detail, more specifically the fascicular interconnections. Median and lingual nerve samples were obtained from five euthanized piglets. OPT images of the samples were acquired and 3D reconstruction was performed. The OPT technique revealed the inner structure of the nerves at high resolution, including large and small fascicles, perineurium, interfascicular tissue, and epineurium. The fascicles were loosely packed inside the median nerve and more densely so in the lingual nerve. Analysis of the 3D models demonstrated that the nerve fascicles can show six general spatial patterns. Fascicular interconnections were clearly identified. The 3D reconstruction of nerve fascicles from OPT images opens a new path for research into the microstructure of the inner contents of fascicular nerve groups and their spatial disposition within the nerve including their interconnections. These techniques enable 3D images of partial areas of nerves to be produced and could became an excellent tool for obtaining data concerning the 3D microanatomy of nerves, essential for better interpretation of ultrasound images in clinical practice and thus avoiding possible neurological complications. Clin. Anat. 31:424-431, 2018. © 2017 Wiley Periodicals, Inc.

Comparison of different tissue clearing methods and 3D imaging techniques for visualization of GFP-expressing mouse embryos and embryonic hearts.

Our goal was to find an optimal tissue clearing protocol for whole-mount imaging of embryonic and adult hearts and whole embryos of transgenic mice that would preserve green fluorescent protein GFP fluorescence and permit comparison of different currently available 3D imaging modalities. We tested various published organic solvent- or water-based clearing protocols intended to preserve GFP fluorescence in central nervous system: tetrahydrofuran dehydration and dibenzylether protocol (DBE), SCALE, CLARITY, and CUBIC and evaluated their ability to render hearts and whole embryos transparent. DBE clearing protocol did not preserve GFP fluorescence; in addition, DBE caused considerable tissue-shrinking artifacts compared to the gold standard BABB protocol. The CLARITY method considerably improved tissue transparency at later stages, but also decreased GFP fluorescence intensity. The SCALE clearing resulted in sufficient tissue transparency up to ED12.5; at later stages the useful depth of imaging was limited by tissue light scattering. The best method for the cardiac specimens proved to be the CUBIC protocol, which preserved GFP fluorescence well, and cleared the specimens sufficiently even at the adult stages. In addition, CUBIC decolorized the blood and myocardium by removing tissue iron. Good 3D renderings of whole fetal hearts and embryos were obtained with optical projection tomography and selective plane illumination microscopy, although at resolutions lower than with a confocal microscope. Comparison of five tissue clearing protocols and three imaging methods for study of GFP mouse embryos and hearts shows that the optimal method depends on stage and level of detail required.

A piecewise monotone subgradient algorithm for accurate L¹-TV based registration of physical slices with discontinuities in microscopy.

Image registration tasks are often formulated in terms of minimization of a functional consisting of a data fidelity term penalizing the mismatch between the reference and the target image, and a term enforcing smoothness of shift between neighboring pairs of pixels (a min-sum problem). Most methods for deformable image registration use some form of interpolation between matching control points. The interpolation makes it impossible to account for isolated discontinuities in the deformation field that may appear, e.g., when a physical slice of a microscopy specimen is ruptured by the cutting tool. For registration of neighboring physical slices of microscopy specimens with discontinuities, Janácek proposed an L¹-distance data fidelity term and a total variation (TV) smoothness term, and used a graph-cut (GC) based iterative steepest descent algorithm for minimization. The L¹-TV functional is nonconvex; hence a steepest descent algorithm is not guaranteed to converge to the global minimum. Schlesinger presented transformation of max-sum problems to minimization of a dual quantity called problem power, which is--contrary to the original max-sum functional--convex. Based on Schlesinger's solution to max-sum problems we developed an algorithm for L¹-TV minimization by iterative multi-label steepest descent minimization of the convex dual problem. For Schlesinger's subgradient algorithm we proposed a novel step control heuristics that considerably enhances both speed and accuracy compared with standard step size strategies for subgradient methods. It is shown experimentally that our subgradient scheme achieves consistently better image registration than GC in terms of lower values both of the composite L¹-TV functional, and of its components, i.e., the L¹ distance of the images and the transformation smoothness TV, and yields visually acceptable results even in cases where the GC based algorithm fails. The new algorithm allows easy parallelization and can thus be sped up by running on multi-core graphic processing units.

A perivascular system releasing sirolimus prevented intimal hyperplasia in a rabbit model in a medium-term study.

The main complication of aortocoronary reconstruction with vein grafts is restenosis in the course of time. The aim was to assess the effect of a periadventitial polyester mesh releasing sirolimus on intimal hyperplasia of autologous grafts. We implanted v. jugularis ext. into a. carotis communis in rabbits. The vein graft was either intact, or was wrapped with a pure polyester mesh, or with a sirolimus-releasing mesh. Three and six weeks after surgery, the veins were subjected to standard histological staining and the thicknesses of the tunica intima, the media and the intima-media complex were measured. Wrapping the vein with a mesh releasing sirolimus or with a pure mesh decreased the thickness of the intima in comparison with a vein graft by 73 ± 11% or 73 ± 8% after 3 weeks, and by 73 ± 9% or 59 ± 12% after 6 weeks, respectively. Sirolimus-releasing meshes reduced the thickness of the media by 65 ± 9% and 20 ± 12% after 3 and 6 weeks. The thickness of the intima-media complex in grafts with sirolimus-releasing meshes decreased by 60 ± 6% and 30 ± 13% in comparison with pure PES meshes, after 3 and 6 weeks, respectively. A periadventitial polyester mesh releasing sirolimus has the potential to become an effective device in preventing vein graft restenosis.

Ecology of testate amoebae in the Komorany ponds in the Vltava Basin.

Testate amoeba (TA) assemblages were collected in 2005 from four ponds in Komorany (Prague, Czech Republic). An analysis of seasonal taxonomic variability of TA populations and its correlation with the limnological characteristics of the area (temperature, pH, total organic carbon, nitrogen, phosphorus, heavy metals, etc.) was performed. The predominant genera were Difflugia, Arcella, and Centropyxis. The most significant changes in the TA community occurred between March and July. Arcella genus dominated in March and April; in May, Arcella and Centropyxis genera were present in the same amount; in June, Arcella genus disappeared, and Difflugia genus started to dominate the community. A multivariate redundancy analysis showed statistically significant correlations between the environmental parameters and the composition of the TA community. The results indicate a negative correlation between TA quantities and Ni, Cd, PAH, Mn, As, and Pb. TA were also affected by concentrations of NH4(+), NO3(-), and P, as well as by temperature variations. The observed correlations between the species composition and environmental parameters can be used in paleoecological interpretations of fossil TA communities. Our results also prove the suitability of TA as water quality indicators in urban areas.

Nonrigid registration of CLSM images of physical sections with discontinuous deformations.

When biological specimens are cut into physical sections for three-dimensional (3D) imaging by confocal laser scanning microscopy, the slices may get distorted or ruptured. For subsequent 3D reconstruction, images from different physical sections need to be spatially aligned by optimization of a function composed of a data fidelity term evaluating similarity between the reference and target images, and a regularization term enforcing transformation smoothness. A regularization term evaluating the total variation (TV), which enables the registration algorithm to account for discontinuities in slice deformation (ruptures), while enforcing smoothness on continuously deformed regions, was proposed previously. The function with TV regularization was optimized using a graph-cut (GC) based iterative solution. However, GC may generate visible registration artifacts, which impair the 3D reconstruction. We present an alternative, multilabel TV optimization algorithm, which in the examined samples prevents the artifacts produced by GC. The algorithm is slower than GC but can be sped up several times when implemented in a multiprocessor computing environment. For image pairs with uneven brightness distribution, we introduce a reformulation of the TV-based registration, in which intensity-based data terms are replaced by comparison of salient features in the reference and target images quantified by local image entropies.

Compensation of inhomogeneous fluorescence signal distribution in 2D images acquired by confocal microscopy.

In images acquired by confocal laser scanning microscopy (CLSM), regions corresponding to the same concentration of fluorophores in the specimen should be mapped to the same grayscale levels. However, in practice, due to multiple distortion effects, CLSM images of even homogeneous specimen regions suffer from irregular brightness variations, e.g., darkening of image edges and lightening of the center. The effects are yet more pronounced in images of real biological specimens. A spatially varying grayscale map complicates image postprocessing, e.g., in alignment of overlapping regions of two images and in 3D reconstructions, since measures of similarity usually assume a spatially independent grayscale map. We present a fast correction method based on estimating a spatially variable illumination gain, and multiplying acquired CLSM images by the inverse of the estimated gain. The method does not require any special calibration of reference images since the gain estimate is extracted from the CLSM image being corrected itself. The proposed approach exploits two types of morphological filters: the median filter and the upper Lipschitz cover. The presented correction method, tested on images of both artificial (homogeneous fluorescent layer) and real biological specimens, namely sections of a rat embryo and a rat brain, proved to be very fast and yielded a significant visual improvement.

Testate amoebae examined by confocal and two-photon microscopy: implications for taxonomy and ecophysiology.

Testate amoebae (TA) are a group of free-living protozoa, important in ecology and paleoecology. Testate amoebae taxonomy is mainly based on the morphological features of the shell, as examined by means of light microscopy or (environmental) scanning electron microscopy (SEM/ESEM). We explored the potential applications of confocal laser scanning microscopy (CLSM), two photon excitation microscopy (TPEM), phase contrast, differential interference contrast (DIC Nomarski), and polarization microscopy to visualize TA shells and inner structures of living cells, which is not possible by SEM or environmental SEM. Images captured by CLSM and TPEM were utilized to create three-dimensional (3D) visualizations and to evaluate biovolume inside the shell by stereological methods, to assess the function of TA in ecosystems. This approach broadens the understanding of TA cell and shell morphology, and inner structures including organelles and endosymbionts, with potential implications in taxonomy and ecophysiology.

Analysis and three-dimensional visualization of collagen in artificial scaffolds using nonlinear microscopy techniques.

Extracellularly distributed collagen and chondrocytes seeded in gelatine and poly-ɛ-caprolactone scaffolds are visualized by two-photon excitation microscopy (TPEM) and second-harmonic generation (SHG) imaging in both forward and backward nondescanned modes. Joint application of TPEM and SHG imaging in combination with stereological measurements of collagen enables us not only to take high-resolution 3-D images, but also to quantitatively analyze the collagen volume and a spatial arrangement of cell-collagen-scaffold systems, which was previously impossible. This novel approach represents a powerful tool for the analysis of collagen-containing scaffolds with applications in cartilage tissue engineering.

Volume reconstruction of large tissue specimens from serial physical sections using confocal microscopy and correction of cutting deformations by elastic registration.

A set of methods leading to volume reconstruction of biological specimens larger than the field of view of a confocal laser scanning microscope (CLSM) is presented. Large tissue specimens are cut into thin physical slices and volume data sets are captured from all studied physical slices by CLSM. Overlapping spatial tiles of the same physical slice are stitched in horizontal direction. Image volumes of successive physical slices are linked in axial direction by applying an elastic registration algorithm to compensate for deformations because of cutting the specimen. We present a method enabling us to keep true object morphology using a priori information about the shape and size of the specimen, available from images of the cutting planes captured by a USB light microscope immediately before cutting the specimen by a microtome. The errors introduced by elastic registration are evaluated using a stereological point counting method and the Procrustes distance. Finally, the images are enhanced to compensate for the effect of the light attenuation with depth and visualized by a hardware accelerated volume rendering. Algorithmic steps of the reconstruction, namely elastic registration, object morphology preservation, image enhancement, and volume visualization, are implemented in a new Rapid3D software package. Because confocal microscopes get more and more frequently used in scientific laboratories, the described volume reconstruction may become an easy-to-apply tool to study large biological objects, tissues, and organs in histology, embryology, evolution biology, and developmental biology. In this work, we demonstrate the reconstruction using a postcranial part of a 17-day-old laboratory Wistar rat embryo.

Methods for compensation of the light attenuation with depth of images captured by a confocal microscope.

A confocal laser scanning microscope (CLSM) enables us to capture images from a biological specimen in different depths and obtain a series of precisely registered fluorescent images. However, images captured from deep layers of the specimen may be darker than images from the topmost layers because of light loss distortions. This effect causes difficulties in subsequent analysis of biological objects. We propose a solution using two approaches: either an online method working already during image acquisition or an offline method assisting as a postprocessing step. In the online method, the gain value of a photomultiplier tube of a CLSM is controlled according to the difference of mean image intensities between the reference and currently acquired image. The offline method consists of two stages. In the first stage, a standard histogram maintaining relative frequencies of gray levels and improving brightness and contrast is created from all images in the series. In the second stage, individual image histograms are warped according to this standard histogram. The methods were tested on real confocal image data captured from human placenta and rat skeletal muscle specimens. It was shown that both approaches diminish the light attenuation in images captured from deep layers of the specimen.