An automated analysis of chemotaxis in vitro using a computer-assisted scanning densitometer

The quantitation of chemotaxis in vitro was developed with a computer-assisted scanning densitometer. The method of estimating the number of cells on a filter was based on the photo-reflection from the nuclei of stained cells. Samples obtained from a 48-well micro chemotaxis assembly were successfully analyzed by this method. This assay system could quantitate chemotaxis much faster and more accurately than by cell counting under the microscope. It was sensitive enough to determine the responsiveness of SMCs and fibroblasts to various chemoattractants. This system could be applied to medical and biological screening tests for drugs and clones in laboratories.     

SANE (Structure Assisted NOE Evaluation): An automated model-based approach for NOE assignment

A reliable automated approach for assignment of NOESY spectra would allow more rapid determination of protein structures by NMR. In this paper we describe a semi-automated procedure for complete NOESY assignment (SANE, Structure Assisted NOE Evaluation), coupled to an iterative procedure for NMR structure determination where the user is directly involved. Our method is similar to ARIA [Nilges et al. (1997) J. Mol. Biol., 269, 408–422], but is compatible with the molecular dynamics suites AMBER and DYANA. The method is ideal for systems where an initial model or crystal structure is available, but has also been used successfully for ab initio structure determination. Use of this semi-automated iterative approach assists in the identification of errors in the NOE assignments to short-cut the path to an NMR solution structure.     

A novel validation algorithm allows for automated cell tracking and the extraction of biologically meaningful parameters

Automated microscopy is currently the only method to non-invasively and label-free observe complex multi-cellular processes, such as cell migration, cell cycle, and cell differentiation. Extracting biological information from a time-series of micrographs requires each cell to be recognized and followed through sequential microscopic snapshots. Although recent attempts to automatize this process resulted in ever improving cell detection rates, manual identification of identical cells is still the most reliable technique. However, its tedious and subjective nature prevented tracking from becoming a standardized tool for the investigation of cell cultures. Here, we present a novel method to accomplish automated cell tracking with a reliability comparable to manual tracking. Previously, automated cell tracking could not rival the reliability of manual tracking because, in contrast to the human way of solving this task, none of the algorithms had an independent quality control mechanism; they missed validation. Thus, instead of trying to improve the cell detection or tracking rates, we proceeded from the idea to automatically inspect the tracking results and accept only those of high trustworthiness, while rejecting all other results. This validation algorithm works independently of the quality of cell detection and tracking through a systematic search for tracking errors. It is based only on very general assumptions about the spatiotemporal contiguity of cell paths. While traditional tracking often aims to yield genealogic information about single cells, the natural outcome of a validated cell tracking algorithm turns out to be a set of complete, but often unconnected cell paths, i.e. records of cells from mitosis to mitosis. This is a consequence of the fact that the validation algorithm takes complete paths as the unit of rejection/acceptance. The resulting set of complete paths can be used to automatically extract important biological parameters with high reliability and statistical significance. These include the distribution of life/cycle times and cell areas, as well as of the symmetry of cell divisions and motion analyses. The new algorithm thus allows for the quantification and parameterization of cell culture with unprecedented accuracy. To evaluate our validation algorithm, two large reference data sets were manually created. These data sets comprise more than 320,000 unstained adult pancreatic stem cells from rat, including 2592 mitotic events. The reference data sets specify every cell position and shape, and assign each cell to the correct branch of its genealogic tree. We provide these reference data sets for free use by others as a benchmark for the future improvement of automated tracking methods.     

A model-based approach for detecting coevolving positions in a molecule

We present a new method for detecting coevolving sites in molecules. The method relies on a set of aligned sequences (nucleic acid or protein) and uses Markov models of evolution to map the substitutions that occurred at each site onto the branches of the underlying phylogenetic tree. This mapping takes into account the uncertainty over ancestral states and among-site rate variation. We then build, for each site, a "substitution vector" containing the posterior estimates of the number of substitutions in each branch. The amount of coevolution for a pair of sites is then measured as the Pearson correlation coefficient between the two corresponding substitution vectors and compared to the expectation under the null hypothesis of independence. We applied the method to a 79-species bacterial ribosomal RNA data set, for which extensive structural characterization has been done over the last 30 years. More than 95% of the intramolecular predicted pairs of sites correspond to known interacting site pairs.     

A model-based approach for mining membrane protein crystallization trials

MOTIVATION: Membrane proteins are known to play crucial roles in various cellular functions. Information about their function can be derived from their structure, but knowledge of these proteins is limited, as their structures are difficult to obtain. Crystallization has proved to be an essential step in the determination of macromolecular structure. Unfortunately, the bottleneck is that the crystallization process is quite complex and extremely sensitive to experimental conditions, the selection of which is largely a matter of trial and error. Even under the best conditions, it can take a large amount of time, from weeks to years, to obtain diffraction-quality crystals. Other issues include the time and cost involved in taking multiple trials and the presence of very few positive samples in a wide and largely undetermined parameter space. Therefore, any help in directing scientists' attention to the hot spots in the conceptual crystallization space would lead to increased efficiency in crystallization trials. RESULTS: This work is an application case study on mining membrane protein crystallization trials to predict novel conditions that have a high likelihood of leading to crystallization. We use suitable supervised learning algorithms to model the data-space and predict a novel set of crystallization conditions. Our preliminary wet laboratory results are very encouraging and we believe this work shows great promise. We conclude with a view of the crystallization space that is based on our results, which should prove useful for future studies in this area.     

An angiogenic extract from skeletal muscle stimulates monocyte and endothelial cell chemotaxis in vitro.

The purpose of this study was to determine whether the extraction of skeletal muscle with a combination of ethanol and hydrochloric acid yields a product capable of stimulating angiogenesis. The resulting extract stimulated inflammation in the rabbit corneal assay, which was followed by capillary formation. In order to determine whether the observed angiogenesis was stimulated by a factor(s) acting directly on the endothelial cells versus a factor(s) recruiting macrophages that in turn release factors acting on endothelial cells, the muscle extract was tested for endothelial cell and monocyte chemotaxis activity in vitro. The muscle extract stimulated significant endothelial cell chemotaxis at concentrations between 94 and 750 micrograms of protein/ml and significant monocyte chemotaxis at concentrations between 8 and 75 micrograms of protein/ml. Polyacrylamide gel electrophoresis suggests that basic fibroblast growth factor and transforming growth factor-beta may be present in this acid/ethanol extract of skeletal muscle.     

Strategies for rare-event detection: an approach for automated fetal cell detection in maternal blood.

This article explores the feasibility of the use of automated microscopy and image analysis to detect the presence of rare fetal nucleated red blood cells (NRBCs) circulating in maternal blood. The rationales for enrichment and for automated image analysis for "rare-event" detection are reviewed. We also describe the application of automated image analysis to 42 maternal blood samples, using a protocol consisting of one-step enrichment followed by immunocytochemical staining for fetal hemoglobin (HbF) and FISH for X- and Y-chromosomal sequences. Automated image analysis consisted of multimode microscopy and subsequent visual evaluation of image memories containing the selected objects. The FISH results were compared with the results of conventional karyotyping of the chorionic villi. By use of manual screening, 43% of the slides were found to be positive (>=1 NRBC), with a mean number of 11 NRBCs (range 1-40). By automated microscopy, 52% were positive, with on average 17 NRBCs (range 1-111). There was a good correlation between both manual and automated screening, but the NRBC yield from automated image analysis was found to be superior to that from manual screening (P=.0443), particularly when the NRBC count was >15. Seven (64%) of 11 XY fetuses were correctly diagnosed by FISH analysis of automatically detected cells, and all discrepancies were restricted to the lower cell-count range. We believe that automated microscopy and image analysis reduce the screening workload, are more sensitive than manual evaluation, and can be used to detect rare HbF-containing NRBCs in maternal blood.     

A new approach to assessing the space use behavior of macroinvertebrates by automated video tracking

1. Individual space and resource use are central issues in ecology and conservation. Recent technological advances such as automated tracking techniques are boosting ecological research in this field. However, the development of a robust method to track space and resource use is still challenging for at least one important ecosystem component: motile aquatic macroinvertebrates. The challenges are mostly related to the small body size and rapid movement of many macroinvertebrate species and to light scattering and wave signal interference in aquatic habitats.
2. We developed a video tracking method designed to reliably assess space use behavior among individual aquatic macroinvertebrates under laboratory (microcosm) conditions. The approach involves the use of experimental apparatus integrating a near infrared backlight source, a Plexiglas multi‐patch maze, multiple infrared cameras, and automated video analysis. It allows detection of the position of fast‐moving (~ 3 cm/s) and translucent individuals of small size (~ 5 mm in length, ~1 mg in dry weight) on simulated resource patches distributed over an experimental microcosm (0.08 m²).
3. To illustrate the adequacy of the proposed method, we present a case study regarding the size dependency of space use behavior in the model organism Gammarus insensibilis, focusing on individual patch selection, giving‐up times, and cumulative space used.
4. In the case study, primary data were collected on individual body size and individual locomotory behavior, for example, mean speed, acceleration, and step length. Individual entrance and departure times were recorded for each simulated resource patch in the experimental maze. Individual giving‐up times were found to be characterized by negative size dependency, with patch departure occurring sooner in larger individuals than smaller ones, and individual cumulative space used (treated as the overall surface area of resource patches that individuals visited) was found to scale positively with body size.
5. This approach to studying space use behavior can deepen our understanding of species coexistence, yielding insights into mechanistic models on larger spatial scales, for example, home range, with implications for ecological and evolutionary processes, as well as for the management and conservation of populations and ecosystems. Despite being specifically developed for aquatic macroinvertebrates, this method can also be applied to other small aquatic organisms such as juvenile fish and amphibians.

     

A mixed model-based variance estimator for marginal model analyses of cluster randomized trials

Generalized estimating equations (GEE) are used in the analysis of cluster randomized trials (CRTs) because: 1) the resulting intervention effect estimate has the desired marginal or population-averaged interpretation, and 2) most statistical packages contain programs for GEE. However, GEE tends to underestimate the standard error of the intervention effect estimate in CRTs. In contrast, penalized quasi-likelihood (PQL) estimates the standard error of the intervention effect in CRTs much better than GEE but is used less frequently because: 1) it generates an intervention effect estimate with a conditional, or cluster-specific, interpretation, and 2) PQL is not a part of most statistical packages. We propose taking the variance estimator from PQL and re-expressing it as a sandwich-type estimator that could be easily incorporated into existing GEE packages, thereby making GEE useful for the analysis of CRTs. Using numerical examples and data from an actual CRT, we compare the performance of this variance estimator to others proposed in the literature, and we find that our variance estimator performs as well as or better than its competitors.     

The use of an automated cell tracking system to identify specific cell types competent for regeneration and transformation

Summary Combining stepper-motors for microscope stage movement with a specially designed software program has led to the establishment of an efficient cell tracking system. Cell immobilization, fixed reference points for calibration of target cell positions, and a video recording system complete the cell finder system. Specific cells can be identified (either beforehand or in retrospect), their locations fixed, and subsequent development of the individual cells monitored daily using computer-assisted relocation. In this way, the specific cell type capable of sustained division and regeneration has recently been identified within a low efficiency protoplast system of a recalcitrant species, sugarbeet. These totipotent cells originated from stomatal guard cells. Isolation and purification procedures were then optimized in a directed way to yield millions of guard cell protoplasts (GCPs). Using polyethylene glycol (PEG)-mediated gene transfer and glucuronidase (GUS) activity for transient expression studies proved that GCPs were amenable to transformation. Gene transfer efficiency was high, as was the number of stably transformed plants that can be produced. At present, the optimized procedure yields 600 transgenic individuals per person per year. This number allows for the selection of the best plants with regard to copy number, DNA insert size, gene expression, and field performance. Prospects for future application of the cell finder system will be discussed. Presented as part of the symposium “Early Events in Tissue Culture and Transformation at the Cellular Level” at the 1997 Congress on In Vitro Biology, Washington, DC 14–18 June 1997.     

A model-based approach to selection of tag SNPs

Background  

Single Nucleotide Polymorphisms (SNPs) are the most common type of polymorphisms found in the human genome. Effective genetic association studies require the identification of sets of tag SNPs that capture as much haplotype information as possible. Tag SNP selection is analogous to the problem of data compression in information theory. According to Shannon's framework, the optimal tag set maximizes the entropy of the tag SNPs subject to constraints on the number of SNPs. This approach requires an appropriate probabilistic model. Compared to simple measures of Linkage Disequilibrium (LD), a good model of haplotype sequences can more accurately account for LD structure. It also provides a machinery for the prediction of tagged SNPs and thereby to assess the performances of tag sets through their ability to predict larger SNP sets.     

Evidence for lymphocyte chemotaxis toward monocytes during PHA-induced aggregation in vitro.

An important, early phenomenon during the development of immune cell interactions in vitro is the formation of multicellular aggregates. We have developed a quantitative assay to determine the kinetics of multicellular aggregate formation within a heterotypic population of cells on a flat surface. This assay follows the time rate of change in the value of an aggregation index for cells in undisturbed culture. For an initial, well-separated population of cells, the index is a minimum and remains at this value if the cells do not move and interact. By contrast, for conditions that promote active cell movement followed by interaction, the index value increases with time. The index, which reflects cells' relative spatial distributions, is an "indirect enumeration" of the number of cells within aggregates as a function of time. We used this index to follow the aggregative behavior of a population of freshly isolated human peripheral lymphocytes and monocytes. Previous studies have shown that monocytes are centrally located within aggregates and that lymphocytes move to surround monocytes. In order to test if lymphocyte movements are random or directed prior to interactions with monocytes, we formulated a simple model to describe changes in the expected number of cells in an "idealized aggregate" as a function of time. A comparison of the model curves with curves generated from the changes in the aggregation index shows that the best fit derives from a model that involves directed movement of lymphocytes toward monocytes. These results suggest that monocytes produce a chemoattracting agent for lymphocytes for these experimental conditions.     

Procedures for the automated analyses of proteoglycans and glycosaminoglycans

Methods for the automated analysis of hexose, uronic acid, and protein using the Technicon AutoAnalyzer II have been developed by modifying previously published procedures. A method of separating glucosamine and galactosamine, which is eminently suited to quantitating one in the presence of a large amount of the other, is reported. Procedures that can be recommended for determining the amino acid content and individual neutral sugars of proteoglycans or glycosaminoglycans are also described.     

A model-based approach for the analysis of neuronal information transmission in multi-input and -output systems

We present a new method to characterize multi-input and output neuronal systems using information theory. To obtain a lower bound of transinformation we take three steps: (1) Estimation of the deterministic response to isolate components carrying stimulus information. The deviation of the original response from the deterministic estimate is defined as noise. (2) Coordinate transformation using PCA yields an uncorrelated representation. (3) Partial transinformation values are calculated independently either by Shannon's formula assuming normality or based on density estimation for arbitrary distributions. We investigate the performance of the algorithms using simulated data and discuss suitable parameter settings. The approach allows to evaluate the degree to which stimulus features are encoded. Its potential is illustrated by analyses of neuronal activity in cat primary visual cortex evoked by electrical retina stimulation.     

A self-consistent cell flux expression for simultaneous chemotaxis and contact guidance in tissues

During wound healing, both chemotaxis and contact guidance can contribute to the migration of blood and tissue cells to the wound. In order to understand the wound healing process, we must thus understand how cells respond to both these simultaneous directional cues, which are not necessarily coaligned. Although chemotaxis and contact guidance have been studied individually, the interaction between them has not been addressed. We extend a stochastic cell movement model, developed by Dickinson and Tranquillo (1995) [6] for individual cues, for simultaneous chemotaxis and contact guidance by a two-parameter perturbation analysis in terms of the two associated cues, a chemotactic factor gradient and aligned tissue fibers. We present results from analysis of the first-order perturbation, which includes the cell flux expression heuristically proposed by others, but reveals paradoxical results for other indices of cell movement, such as the mean-squared displacement. We then present second-order perturbation results that resolve these paradoxical results. Finally, we relate these results to a continuum mechanical model developed by Barocas and Tranquillo (1997) [3] that predicts fiber alignment due to cell traction induced tissue contraction. Received: 30 April 1999 / Revised version: 30 October 1999 / Published online: 14 September 2000     

Development of a dual-wavelength fluorescent nanoprobe for in vivo and in vitro cell tracking consecutively

Many imaging probes have been developed for a wide variety of imaging modalities. However, no optical imaging probe could be utilized for both microscopic and whole animal imaging. To fill the gap, the dual-wavelength fluorescent imaging nanoprobe was developed to simultaneously carry both visible-range fluorescent dye and near-infrared (NIR) dye. Emission scan confirms that the nanoprobe exhibits two separate peaks with strong fluorescent intensity in both visible and NIR ranges. Furthermore, the dual-wavelength fluorescent nanoprobe has high photostability and colloidal stability, as well as long shelf-life. In vitro cell culture experiments show that the nanoprobe has the ability to label different types of cells (namely, esophageal, prostate, fibroblast and macrophage cell) for fluorescent microscope imaging. More importantly, cell tracking experiments confirm that cell migration and distribution in various organs can be tracked in real time using in vivo whole-body NIR imaging and in vitro microscopic imaging, respectively.     

Using light to shape chemical gradients for parallel and automated analysis of chemotaxis

Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96‐well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 siRNA perturbations. A key finding was that the G‐protein G_iα subunit selectively controls the direction of migration while the receptor and Gβ subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP. The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low‐ and high‐resolution fluorescence microscopy.     

A second-generation device for automated training and quantitative behavior analyses of molecularly-tractable model organisms

A deep understanding of cognitive processes requires functional, quantitative analyses of the steps leading from genetics and the development of nervous system structure to behavior. Molecularly-tractable model systems such as Xenopus laevis and planaria offer an unprecedented opportunity to dissect the mechanisms determining the complex structure of the brain and CNS. A standardized platform that facilitated quantitative analysis of behavior would make a significant impact on evolutionary ethology, neuropharmacology, and cognitive science. While some animal tracking systems exist, the available systems do not allow automated training (feedback to individual subjects in real time, which is necessary for operant conditioning assays). The lack of standardization in the field, and the numerous technical challenges that face the development of a versatile system with the necessary capabilities, comprise a significant barrier keeping molecular developmental biology labs from integrating behavior analysis endpoints into their pharmacological and genetic perturbations. Here we report the development of a second-generation system that is a highly flexible, powerful machine vision and environmental control platform. In order to enable multidisciplinary studies aimed at understanding the roles of genes in brain function and behavior, and aid other laboratories that do not have the facilities to undergo complex engineering development, we describe the device and the problems that it overcomes. We also present sample data using frog tadpoles and flatworms to illustrate its use. Having solved significant engineering challenges in its construction, the resulting design is a relatively inexpensive instrument of wide relevance for several fields, and will accelerate interdisciplinary discovery in pharmacology, neurobiology, regenerative medicine, and cognitive science.     

Studies of bacterial chemotaxis in defined concentration gradients. A model for chemotaxis toward L-serine

The details of the chemotactic response of Salmonella typhimurium to gradients of L-serine have been examined in some detail. Two relatively macroscopic techniques have been employed to measure the bacterial response. These include measurements of the average velocity as the bacterial population moves toward attractants, and measurement of the upward-to-downward flux ratio, R, in the stable preformed attractant gradients. The dependence of the average velocity on gradient appears to be hyperbolic in nature, while the flux ratio depends linearly on the gradient. These data suggest a microscopic model for the dependence of bacterial behavior on the serine gradient. The model involves a linear dependence of the mean lifetime of a bacterial trajectory on the gradient for those bacteria moving toward higher attractant concentration. Those moving toward low concentrations of attractant do not change the mean duration of their trajectories, or the speed at which a given bacterium swims through the solution. This model generates the observed dependences of the average velocity and flux ratio on gradient. Interpretation of the experimental data suggests that a gradient which increases serine concentration by a factor of 2 in 10 mm is sufficient to double the average duration of a trajectory for a bacterium moving directly up the gradient. The concentration dependence of the chemotactic response to serine is more complicated. It suggests that more than one receptor of serine may be involved in determining chemotactic behavior to this attractant.     

A hybrid approach for the automated finishing of bacterial genomes

Advances in DNA sequencing technology have improved our ability to characterize most genomic diversity. However, accurate resolution of large structural events is challenging because of the short read lengths of second-generation technologies. Third-generation sequencing technologies, which can yield longer multikilobase reads, have the potential to address limitations associated with genome assembly. Here we combine sequencing data from second- and third-generation DNA sequencing technologies to assemble the two-chromosome genome of a recent Haitian cholera outbreak strain into two nearly finished contigs at >99.9% accuracy. Complex regions with clinically relevant structure were completely resolved. In separate control assemblies on experimental and simulated data for the canonical N16961 cholera reference strain, we obtained 14 scaffolds of greater than 1 kb for the experimental data and 8 scaffolds of greater than 1 kb for the simulated data, which allowed us to correct several errors in contigs assembled from the short-read data alone. This work provides a blueprint for the next generation of rapid microbial identification and full-genome assembly.