Reproducible image handling and analysis.

Image data are universal in life sciences research. Their proper handling is not. A significant proportion of image data in research papers show signs of mishandling that undermine their interpretation. We propose that a precise description of the image processing and analysis applied is required to address this problem. A new norm for reporting reproducible image analyses will diminish mishandling, as it will alert co-authors, referees, and journals to aberrant image data processing or, if published nonetheless, it will document it to the reader. To promote this norm, we discuss the effectiveness of this approach and give some step-by-step instructions for publishing reproducible image data processing and analysis workflows.

Posttranscriptional Regulation of the Human LDL Receptor by the U2-Spliceosome.

BACKGROUND: The LDLR (low-density lipoprotein receptor) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. METHODS: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of data sets on gene expression and variants in human populations. RESULTS: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR. The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in nontransfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in nonalcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and 3 rare variants of one spliceosome gene, RBM25, are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared with overexpression of wild-type RBM25, overexpression of the 3 rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. CONCLUSIONS: We identified a novel mechanism of posttranscriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.

AutoTube: a novel software for the automated morphometric analysis of vascular networks in tissues.

Due to their involvement in many physiologic and pathologic processes, there is a great interest in identifying new molecular pathways that mediate the formation and function of blood and lymphatic vessels. Vascular research increasingly involves the image-based analysis and quantification of vessel networks in tissue whole-mounts or of tube-like structures formed by cultured endothelial cells in vitro. While both types of experiments deliver important mechanistic insights into (lymph)angiogenic processes, the manual analysis and quantification of such experiments are typically labour-intensive and affected by inter-experimenter variability. To bypass these problems, we developed AutoTube, a new software that quantifies parameters like the area covered by vessels, vessel width, skeleton length and branching or crossing points of vascular networks in tissues and in in vitro assays. AutoTube is freely downloadable, comprises an intuitive graphical user interface and helps to perform otherwise highly time-consuming image analyses in a rapid, automated and reproducible manner. By analysing lymphatic and blood vascular networks in whole-mounts prepared from different tissues or from gene-targeted mice with known vascular abnormalities, we demonstrate the ability of AutoTube to determine vascular parameters in close agreement to the manual analyses and to identify statistically significant differences in vascular morphology in tissues and in vascular networks formed in in vitro assays.

How Modifications of Corneal Cross-Linking Protocols Influence Corneal Resistance to Enzymatic Digestion and Treatment Depth.

Purpose: The purpose of this study was to determine the effects of the Photoactivated Chromophore for Keratitis Corneal Cross-Linking (PACK-CXL) protocol modifications on corneal resistance to enzymatic digestion and treatment depth. Methods: Eight hundred one ex vivo porcine eyes were randomly divided into groups of 12 to 86 corneas, treated with various epi-off PACK-CXL modifications, including acceleration (30 > 2 minutes, 5.4 J/cm2), increased fluence (5.4 > 32.4 J/cm2), deuterium oxide (D2O) supplementation, different carrier types (dextran versus hydroxypropyl methylcellulose [HPMC]), increased riboflavin concentration (0.1 > 0.4%), and riboflavin replenishment during irradiation (yes/no). Control group eyes did not receive PACK-CXL. A pepsin digestion assay was used to determine corneal resistance to enzymatic digestion. A phalloidin fluorescent imaging assay was used to determine the PACK-CXL treatment effect depth. Differences between groups were evaluated using a linear model and a derivative method, respectively. Results: PACK-CXL significantly increased corneal resistance to enzymatic digestion compared to no treatment (P < 0.03). When compared to a 10 minute, 5.4 J/cm2 PACK-CXL protocol, fluences of 16.2 J/cm2 and higher increased corneal resistance to enzymatic digestion by 1.5- to 2-fold (P < 0.001). Other protocol modifications did not significantly change corneal resistance. A 16.2 J/cm2 fluence also increased collagen compaction in the anterior stroma, whereas omitting riboflavin replenishment during irradiation increased PACK-CXL treatment depth. Conclusions: Increasing fluence will likely optimize PACK-CXL treatment effectiveness. Treatment acceleration reduces treatment duration without compromising effectiveness. Translational Relevance: The generated data help to optimize clinical PACK-CXL settings and direct future research efforts.

RNAi Screening Uncovers a Synthetic Sick Interaction between CtIP and the BARD1 Tumor Suppressor.

Human CtIP is best known for its role in DNA end resection to initiate DNA double-strand break repair by homologous recombination. Recently, CtIP has also been shown to protect reversed replication forks from nucleolytic degradation upon DNA replication stress. However, still little is known about the DNA damage response (DDR) networks that preserve genome integrity and sustain cell survival in the context of CtIP insufficiency. Here, to reveal such potential buffering relationships, we screened a DDR siRNA library in CtIP-deficient cells to identify candidate genes that induce synthetic sickness/lethality (SSL). Our analyses unveil a negative genetic interaction between CtIP and BARD1, the heterodimeric binding partner of BRCA1. We found that simultaneous disruption of CtIP and BARD1 triggers enhanced apoptosis due to persistent replication stress-induced DNA lesions giving rise to chromosomal abnormalities. Moreover, we observed that the genetic interaction between CtIP and BARD1 occurs independently of the BRCA1-BARD1 complex formation and might be, therefore, therapeutical relevant for the treatment of BRCA-defective tumors.

The NEUBIAS Gateway: a hub for bioimage analysis methods and materials.

We introduce the NEUBIAS Gateway, a new platform for publishing materials related to bioimage analysis, an interdisciplinary field bridging computer science and life sciences. This emerging field has been lacking a central place to share the efforts of the growing group of scientists addressing biological questions using image data. The Gateway welcomes a wide range of publication formats including articles, reviews, reports and training materials. We hope the Gateway further supports this important field to grow and helps more biologists and computational scientists learn about and contribute to these efforts.

Bidirectional Propagation of Signals and Nutrients in Fungal Networks via Specialized Hyphae.

Intercellular distribution of nutrients and coordination of responses to internal and external cues via endogenous signaling molecules are hallmarks of multicellular organisms. Vegetative mycelia of multicellular fungi are syncytial networks of interconnected hyphae resulting from hyphal tip growth, branching, and fusion. Such mycelia can reach considerable dimensions and, thus, different parts can be exposed to quite different environmental conditions. Our knowledge about the mechanisms by which fungal mycelia can adjust nutrient gradients or coordinate their defense response to fungivores is scarce, in part due to limitations in technologies currently available for examining different parts of a mycelium over longer time periods at the microscopic level. Here, we combined a tailor-made microfluidic platform with time-lapse fluorescence microscopy to visualize the dynamic response of the vegetative mycelium of a basidiomycete to two different stimuli. The microfluidic platform allows simultaneous monitoring at both the colony and single-hypha level. We followed the dynamics of the distribution of a locally administered nutrient analog and the defense response to spatially confined predation by a fungivorous nematode. Although both responses of the mycelium were constrained locally, we observed long-distance propagation for both the nutrient analog and defense response in a subset of hyphae. This propagation along hyphae occurred in both acropetal and basipetal directions and, intriguingly, the direction was found to alternate every 3 hr in an individual hypha. These results suggest that multicellular fungi have, as of yet, undescribed mechanisms to coordinate the distribution of nutrients and their behavioral response upon attack by fungivores.

Quantitative spatial analysis of haematopoiesis-regulating stromal cells in the bone marrow microenvironment by 3D microscopy.

Sinusoidal endothelial cells and mesenchymal CXCL12-abundant reticular cells are principal bone marrow stromal components, which critically modulate haematopoiesis at various levels, including haematopoietic stem cell maintenance. These stromal subsets are thought to be scarce and function via highly specific interactions in anatomically confined niches. Yet, knowledge on their abundance, global distribution and spatial associations remains limited. Using three-dimensional quantitative microscopy we show that sinusoidal endothelial and mesenchymal reticular subsets are remarkably more abundant than estimated by conventional flow cytometry. Moreover, both cell types assemble in topologically complex networks, associate to extracellular matrix and pervade marrow tissues. Through spatial statistical methods we challenge previous models and demonstrate that even in the absence of major specific interaction forces, virtually all tissue-resident cells are invariably in physical contact with, or close proximity to, mesenchymal reticular and sinusoidal endothelial cells. We further show that basic structural features of these stromal components are preserved during ageing.

Self-organized critical system with no stationary attractor state.

A simple model economy with interacting producers and consumers is introduced. When driven by extreme dynamics, the model self-organizes not to an attractor state, but to an asymptote, on which the economy has a constant rate of deflation, is critical, and exhibits avalanches of activity with power-law distributed sizes. This example demonstrates that self-organized critical behavior occurs in a larger class of systems than so far considered: systems not driven to an attractive fixed point, but, e.g., an asymptote, may also display self-organized criticality.

Harmonic oscillator in heat bath: exact simulation of time-lapse-recorded data and exact analytical benchmark statistics.

The stochastic dynamics of the damped harmonic oscillator in a heat bath is simulated with an algorithm that is exact for time steps of arbitrary size. Exact analytical results are given for correlation functions and power spectra in the form they acquire when computed from experimental time-lapse recordings. Three applications are discussed: (i) The effects of finite sampling rate and time, described exactly here, are similar for other stochastic dynamical systems--e.g., motile microorganisms and their time-lapse-recorded trajectories. (ii) The same statistics is satisfied by any experimental system to the extent that it is interpreted as a damped harmonic oscillator at finite temperature-such as an AFM cantilever. (iii) Three other models of fundamental interest are limiting cases of the damped harmonic oscillator at finite temperature; it consequently bridges their differences and describes the effects of finite sampling rate and sampling time for these models as well.

Power spectrum analysis with least-squares fitting: amplitude bias and its elimination, with application to optical tweezers and atomic force microscope cantilevers.

Optical tweezers and atomic force microscope (AFM) cantilevers are often calibrated by fitting their experimental power spectra of Brownian motion. We demonstrate here that if this is done with typical weighted least-squares methods, the result is a bias of relative size between -2/n and +1/n on the value of the fitted diffusion coefficient. Here, n is the number of power spectra averaged over, so typical calibrations contain 10%-20% bias. Both the sign and the size of the bias depend on the weighting scheme applied. Hence, so do length-scale calibrations based on the diffusion coefficient. The fitted value for the characteristic frequency is not affected by this bias. For the AFM then, force measurements are not affected provided an independent length-scale calibration is available. For optical tweezers there is no such luck, since the spring constant is found as the ratio of the characteristic frequency and the diffusion coefficient. We give analytical results for the weight-dependent bias for the wide class of systems whose dynamics is described by a linear (integro)differential equation with additive noise, white or colored. Examples are optical tweezers with hydrodynamic self-interaction and aliasing, calibration of Ornstein-Uhlenbeck models in finance, models for cell migration in biology, etc. Because the bias takes the form of a simple multiplicative factor on the fitted amplitude (e.g. the diffusion coefficient), it is straightforward to remove and the user will need minimal modifications to his or her favorite least-squares fitting programs. Results are demonstrated and illustrated using synthetic data, so we can compare fits with known true values. We also fit some commonly occurring power spectra once-and-for-all in the sense that we give their parameter values and associated error bars as explicit functions of experimental power-spectral values.

Persistent cell motion in the absence of external signals: a search strategy for eukaryotic cells.

BACKGROUND: Eukaryotic cells are large enough to detect signals and then orient to them by differentiating the signal strength across the length and breadth of the cell. Amoebae, fibroblasts, neutrophils and growth cones all behave in this way. Little is known however about cell motion and searching behavior in the absence of a signal. Is individual cell motion best characterized as a random walk? Do individual cells have a search strategy when they are beyond the range of the signal they would otherwise move toward? Here we ask if single, isolated, Dictyostelium and Polysphondylium amoebae bias their motion in the absence of external cues. METHODOLOGY: We placed single well-isolated Dictyostelium and Polysphondylium cells on a nutrient-free agar surface and followed them at 10 sec intervals for approximately 10 hr, then analyzed their motion with respect to velocity, turning angle, persistence length, and persistence time, comparing the results to the expectation for a variety of different types of random motion. CONCLUSIONS: We find that amoeboid behavior is well described by a special kind of random motion: Amoebae show a long persistence time ( approximately 10 min) beyond which they start to lose their direction; they move forward in a zig-zag manner; and they make turns every 1-2 min on average. They bias their motion by remembering the last turn and turning away from it. Interpreting the motion as consisting of runs and turns, the duration of a run and the amplitude of a turn are both found to be exponentially distributed. We show that this behavior greatly improves their chances of finding a target relative to performing a random walk. We believe that other eukaryotic cells may employ a strategy similar to Dictyostelium when seeking conditions or signal sources not yet within range of their detection system.

Surface forces and drag coefficients of microspheres near a plane surface measured with optical tweezers.

Optical tweezers are widely used to measure molecular forces in biology. Such measurements are often influenced by a nearby surface that can perturb both the calibration of the tweezers as well as the hydrodynamic forces acting on microspheres to which the biomolecules are attached. In this study, we have used a very stable optical tweezers setup employing a recently developed calibration method (Tolic-Nørrelykke, S. F.; Schäffer, E.; Howard, J.; Pavone, F. S.; Jülicher, F.; Flyvbjerg, H. Rev. Sci. Instrum. 2006, 77 (10), 103101) to determine how the calibration of the tweezers and the forces on the microspheres depend on the height above the surface. We show that the displacement sensitivity of the tweezers is modulated by a standing light wave between the microsphere and the surface. We measured the dependence of the drag coefficient on height and compared it to exact and closed-form solutions to the Navier-Stokes equations. Also, we measured the surface force gradients in different salt solutions and for different surface blocking methods. For a given blocking method, our data suggest that microspheres can experience attractive and/or repulsive forces close to surfaces. For example, a Teflon layer reduces attractive interactions, and the presence of casein can lead to long-range repulsive interactions. These measurements are a prerequisite for the accurate measurement of normal forces with respect to an interface that occur in biological molecules held between surfaces.

Stepwise bending of DNA by a single TATA-box binding protein.

The TATA-box binding protein (TBP) is required by all three eukaryotic RNA polymerases for the initiation of transcription from most promoters. TBP recognizes, binds to, and bends promoter sequences called "TATA-boxes" in the DNA. We present results from the study of individual Saccharomyces cerevisiae TBPs interacting with single DNA molecules containing a TATA-box. Using video microscopy, we observed the Brownian motion of beads tethered by short surface-bound DNA. When TBP binds to and bends the DNA, the conformation of the DNA changes and the amplitude of Brownian motion of the tethered bead is reduced compared to that of unbent DNA. We detected individual binding and dissociation events and derived kinetic parameters for the process. Dissociation was induced by increasing the salt concentration or by directly pulling on the tethered bead using optical tweezers. In addition to the well-defined free and bound classes of Brownian motion, we observed another two classes of motion. These extra classes were identified with intermediate states on a three-step, linear-binding pathway. Biological implications of the intermediate states are discussed.

Diversity in the rates of transcript elongation by single RNA polymerase molecules.

Single-molecule measurements of the activities of a variety of enzymes show that rates of catalysis may vary markedly between different molecules in putatively homogeneous enzyme preparations. We measured the rate at which purified Escherichia coli RNA polymerase moves along a approximately 2650-bp DNA during transcript elongation in vitro at 0.5 mm nucleoside triphosphates. Individual molecules of a specifically biotinated RNA polymerase derivative were tagged with 199-nm diameter avidin-coated polystyrene beads; enzyme movement along a surface-linked DNA molecule was monitored by observing changes in bead Brownian motion by light microscopy. The DNA was derived from a naturally occurring transcription unit and was selected for the absence of regulatory sequences that induce lengthy pausing or termination of transcription. With rare exceptions, individual enzyme molecules moved at a constant velocity throughout the transcription reaction; the distribution of velocities across a population of 140 molecules was unimodal and was well fit by a Gaussian. However, the width of the Gaussian, sigma = 6.7 bp/s, was considerably larger than the precision of the velocity measurement (1 bp/s). The observations show that different transcription complexes have differences in catalytic rate (and thus differences in structure) that persist for thousands of catalytic turnovers. These differences may provide a parsimonious explanation for the complex transcription kinetics observed in bulk solution.