Detecting the effect of genetic diversity on brain composition in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is broadly characterized by neurodegeneration, pathology accumulation, and cognitive decline. There is considerable variation in the progression of clinical symptoms and pathology in humans, highlighting the importance of genetic diversity in the study of AD. To address this, we analyze cell composition and amyloid-beta deposition of 6- and 14-month-old AD-BXD mouse brains.

Detecting the effect of genetic diversity on brain composition in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is characterized by neurodegeneration, pathology accumulation, and progressive cognitive decline. There is significant variation in age at onset and severity of symptoms highlighting the importance of genetic diversity in the study of AD. To address this, we analyzed cell and pathology composition of 6- and 14-month-old AD-BXD mouse brains using the semi-automated workflow (QUINT); which we expanded to allow for nonlinear refinement of brain atlas-registration, and quality control assessment of atlas-registration and brain section integrity.

3D Atlas of the Pituitary Gland of the Model Fish Medaka ().

In vertebrates, the anterior pituitary plays a crucial role in regulating several essential physiological processes the secretion of at least seven peptide hormones by different endocrine cell types. Comparative and comprehensive knowledge of the spatial distribution of those endocrine cell types is required to better understand their physiological functions. Using medaka as a model and several combinations of multi-color fluorescence hybridization, we present the first 3D atlas revealing the gland-wide distribution of seven endocrine cell populations: lactotropes, thyrotropes, Lh and Fsh gonadotropes, somatotropes, and -expressing cells (corticotropes and melanotropes) in the anterior pituitary of a teleost fish.

An Easy Path for Correlative Electron and Super-Resolution Light Microscopy.

A number of new Correlative Light and Electron Microscopy approaches have been developed over the past years, offering the opportunity to combine the specificity and bio-compatibility of light microscopy with the high resolution achieved in electron microscopy. More recently, these approaches have taken one step further and also super-resolution light microscopy was combined with transmission or scanning electron microscopy. This combination usually requires moving the specimen between different imaging systems, an expensive set-up and relatively complicated imaging workflows.

Spatial registration of serial microscopic brain images to three-dimensional reference atlases with the QuickNII tool.

Modern high throughput brain wide profiling techniques for cells and their morphology, connectivity, and other properties, make the use of reference atlases with 3D coordinate frameworks essential. However, anatomical location of observations made in microscopic sectional images from rodent brains is typically determined by comparison with 2D anatomical reference atlases. A major challenge in this regard is that microscopic sections often are cut with orientations deviating from the standard planes used in the reference atlases, resulting in inaccuracies and a need for tedious correction steps.

Apoptosis and eryptosis: Striking differences on biomembrane level.

The cell plasma membrane plays an essential role in programmed cell death of nucleated cells (apoptosis) and erythrocytes (eryptosis), and its changes due to loss of transmembrane asymmetry are quite similar. However, nucleated cells possess the network of intracellular membranes, which are missing in erythrocytes. Providing comparative studies with series of molecular probes, we observe dramatic differences in membrane lipid order in the course of apoptosis and eryptosis.

Brain-Wide Mapping of Axonal Connections: Workflow for Automated Detection and Spatial Analysis of Labeling in Microscopic Sections.

Axonal tracing techniques are powerful tools for exploring the structural organization of neuronal connections. Tracers such as biotinylated dextran amine (BDA) and Phaseolus vulgaris leucoagglutinin (Pha-L) allow brain-wide mapping of connections through analysis of large series of histological section images. We present a workflow for efficient collection and analysis of tract-tracing datasets with a focus on newly developed modules for image processing and assignment of anatomical location to tracing data.

Genome-wide RNAi Screening Identifies Protein Modules Required for 40S Subunit Synthesis in Human Cells.

Ribosome biogenesis is a highly complex process requiring many assisting factors. Studies in yeast have yielded comprehensive knowledge of the cellular machinery involved in this process. However, many aspects of ribosome synthesis are different in higher eukaryotes, and the global set of mammalian ribosome biogenesis factors remains unexplored.

CIDRE: an illumination-correction method for optical microscopy.

Uneven illumination affects every image acquired by a microscope. It is often overlooked, but it can introduce considerable bias to image measurements. The most reliable correction methods require special reference images, and retrospective alternatives do not fully model the correction process.

Simultaneous analysis of large-scale RNAi screens for pathogen entry.

Background: Large-scale RNAi screening has become an important technology for identifying genes involved in biological processes of interest. However, the quality of large-scale RNAi screening is often deteriorated by off-targets effects. In order to find statistically significant effector genes for pathogen entry, we systematically analyzed entry pathways in human host cells for eight pathogens using image-based kinome-wide siRNA screens with siRNAs from three vendors.

Experimenting liver fibrosis diagnostic by two photon excitation microscopy and Bag-of-Features image classification.

The accurate staging of liver fibrosis is of paramount importance to determine the state of disease progression, therapy responses, and to optimize disease treatment strategies. Non-linear optical microscopy techniques such as two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) can image the endogenous signals of tissue structures and can be used for fibrosis assessment on non-stained tissue samples. While image analysis of collagen in SHG images was consistently addressed until now, cellular and tissue information included in TPEF images, such as inflammatory and hepatic cell damage, equally important as collagen deposition imaged by SHG, remain poorly exploited to date.

Nuclear motility in glioma cells reveals a cell-line dependent role of various cytoskeletal components.

Nuclear migration is a general term for the movement of the nucleus towards a specific site in the cell. These movements are involved in a number of fundamental biological processes, such as fertilization, cell division, and embryonic development. Despite of its importance, the mechanism of nuclear migration is still poorly understood in mammalian cells.

RNAiAtlas: a database for RNAi (siRNA) libraries and their specificity.

Large-scale RNA interference (RNAi) experiments, especially the ones based on short-interfering RNA (siRNA) technology became increasingly popular over the past years. For such knock-down/screening purposes, different companies offer sets of oligos/reagents targeting the whole genome or a subset of it for various organisms. Obviously, the sequence (and structure) of the corresponding oligos is a key factor in obtaining reliable results in these large-scale studies and the companies use a variety of (often not fully public) algorithms to design them.

Machine learning improves the precision and robustness of high-content screens: using nonlinear multiparametric methods to analyze screening results.

Imaging-based high-content screens often rely on single cell-based evaluation of phenotypes in large data sets of microscopic images. Traditionally, these screens are analyzed by extracting a few image-related parameters and use their ratios (linear single or multiparametric separation) to classify the cells into various phenotypic classes. In this study, the authors show how machine learning-based classification of individual cells outperforms those classical ratio-based techniques.

Acquisition speed comparison of microscope software programs.

Reliable software is a prerequisite for successful operation of a modern wide field fluorescence microscope. When used for live cell imaging, acquisition speed is of particular interest. This is both because biological processes can be highly-dynamic, and to avoid unnecessary photobleaching and phototoxicity of living samples.

A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export.

The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits.

Kernelized Z' factor in multiparametric screening technology.

RNA interference (RNAi) high-content screening (HCS) enables massive parallel gene silencing and is increasingly being used to reveal novel connections between genes and disease-relevant phenotypes. The application of genome-scale RNAi relies on the development of high quality HCS assays. The Z' factor statistic provides a way to evaluate whether or not screening run conditions (reagents, protocols, instrumentation, kinetics, and other conditions not directly related to the test compounds) are optimized.

Workflow-based software environment for large-scale biological experiments.

High-content screening (HCS) technologies are becoming increasingly used in both large-scale drug discovery and basic research programs. These automated imaging and analysis technologies enable the researcher to elucidate the complex biology that underlies the functions of genes, proteins, and other biomolecules at the cellular level. HCS combines the power of automated digital microscopy and advanced software-based image analysis algorithms to detect and quantify biological changes in cells and tissues.

Automated suppression of sample-related artifacts in Fluorescence Correlation Spectroscopy.

Fluorescence Correlation Spectroscopy (FCS) in cells often suffers from artifacts caused by bright aggregates or vesicles, depletion of fluorophores or bleaching of a fluorescent background. The common practice of manually discarding distorted curves is time consuming and subjective. Here we demonstrate the feasibility of automated FCS data analysis with efficient rejection of corrupted parts of the signal.

A classical NLS and the SUN domain contribute to the targeting of SUN2 to the inner nuclear membrane.

Integral membrane proteins of the inner nuclear membrane (INM) are inserted into the endoplasmic reticulum membrane during their biogenesis and are then targeted to their final destination. We have used human SUN2 to delineate features that are required for INM targeting and have identified multiple elements that collectively contribute to the efficient localization of SUN2 to the nuclear envelope (NE). One such targeting element is a classical nuclear localization signal (cNLS) present in the N-terminal, nucleoplasmic domain of SUN2.

Locomotion of fish epidermal keratocytes on spatially selective adhesion patterns.

Cell migration results from forces generated by assembly, contraction, and adhesion of the cytoskeleton. To address how these forces integrate in space and time, novel assays are required that allow spatial separation of the different force categories. We used micro-contact printing of fibronectin on glass substrates to study the effect of adhesion patterns on fish epidermal keratocytes locomotion.

Surface engineering approaches to micropattern surfaces for cell-based assays.

The ability to produce patterns of single or multiple cells through precise surface engineering of cell culture substrates has promoted the development of cellular bioassays that provide entirely new insights into the factors that control cell adhesion to material surfaces, cell proliferation, differentiation and molecular signaling pathways. The ability to control shape and spreading of attached cells and cell-cell contacts through the form and dimension of the cell-adhesive patches with high precision is important. Commitment of stem cells to different specific lineages depends strongly on cell shape, implying that controlled microenvironments through engineered surfaces may not only be a valuable approach towards fundamental cell-biological studies, but also of great importance for the design of cell culture substrates for tissue engineering.

Tocopherol cyclase (VTE1) localization and vitamin E accumulation in chloroplast plastoglobule lipoprotein particles.

Chloroplasts contain lipoprotein particles termed plastoglobules. Plastoglobules are generally believed to have little function beyond lipid storage. Here we report on the identification of plastoglobule proteins using mass spectrometry methods in Arabidopsis thaliana.

Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers.

Expression of alpha-smooth muscle actin (alpha-SMA) renders fibroblasts highly contractile and hallmarks myofibroblast differentiation. We identify alpha-SMA as a mechanosensitive protein that is recruited to stress fibers under high tension. Generation of this threshold tension requires the anchoring of stress fibers at sites of 8-30-microm-long "supermature" focal adhesions (suFAs), which exert a stress approximately fourfold higher (approximately 12 nN/microm2) on micropatterned deformable substrates than 2-6-microm-long classical FAs.