The curvHDR Method for Gating Flow Cytometry Samples

Motivation: High-throughput flow cytometry experiments produce hundreds of large multivariate samples of cellular characteristics. These samples require specialized processing to obtain clinically meaningful measurements. A major component of this processing is a form of cell subsetting known as gating. Manual gating is time-consuming and subjective. Good automatic and semi-automatic gating algorithms are very beneficial to high-throughput flow cytometry. Results: We develop a statistical procedure, named curvHDR, for automatic and semi-automatic gating. The method combines the notions of significant high negative curvature regions and highest density regions and has the ability to adapt well to human-perceived gates. The underlying principles apply to dimension of arbitrary size, although we focus on dimensions up to three. Accompanying software, compatible with contemporary flow cytometry informatics, is developed. Availability: Software for Bioconductor within R is available

flowCore: a Bioconductor package for high throughput flow cytometry

<p>Abstract</p> <p>Background</p> <p>Recent advances in automation technologies have enabled the use of flow cytometry for high throughput screening, generating large complex data sets often in clinical trials or drug discovery settings. However, data management and data analysis methods have not advanced sufficiently far from the initial small-scale studies to support modeling in the presence of multiple covariates.</p> <p>Results</p> <p>We developed a set of flexible open source computational tools in the R package flowCore to facilitate the analysis of these complex data. A key component of which is having suitable data structures that support the application of similar operations to a collection of samples or a clinical cohort. In addition, our software constitutes a shared and extensible research platform that enables collaboration between bioinformaticians, computer scientists, statisticians, biologists and clinicians. This platform will foster the development of novel analytic methods for flow cytometry.</p> <p>Conclusion</p> <p>The software has been applied in the analysis of various data sets and its data structures have proven to be highly efficient in capturing and organizing the analytic work flow. Finally, a number of additional Bioconductor packages successfully build on the infrastructure provided by flowCore, open new avenues for flow data analysis.</p

Computational analysis of microbial flow cytometry data

Flow cytometry is an important technology for the study of microbial communities. It grants the ability to rapidly generate phenotypic single-cell data that are both quantitative, multivariate and of high temporal resolution. The complexity and amount of data necessitate an objective and streamlined data processing workflow that extends beyond commercial instrument software. No full overview of the necessary steps regarding the computational analysis of microbial flow cytometry data currently exists. In this review, we provide an overview of the full data analysis pipeline, ranging from measurement to data interpretation, tailored toward studies in microbial ecology. At every step, we highlight computational methods that are potentially useful, for which we provide a short nontechnical description. We place this overview in the context of a number of open challenges to the field and offer further motivation for the use of standardized flow cytometry in microbial ecology research

BMC Bioinformatics

BackgroundHigh-throughput technologies such as flow and mass cytometry have the potential to illuminate cellular networks. However, analyzing the data produced by these technologies is challenging. Visualization is needed to help researchers explore this data.ResultsWe developed a web-based software program, NetworkPainter, to enable researchers to analyze dynamic cytometry data in the context of pathway diagrams. NetworkPainter provides researchers a graphical interface to draw and \ue2\u20ac\u153paint\ue2\u20ac? pathway diagrams with experimental data, producing animated diagrams which display the activity of each network node at each time point.ConclusionNetworkPainter enables researchers to more fully explore multi-parameter, dynamical cytometry data.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0602-4) contains supplementary material, which is available to authorized users.1P50GM107615/GM/NIGMS NIH HHS/United States5DP1LM01150-05/DP/NCCDPHP CDC HHS/United StatesCA125994-01A1/CA/NCI NIH HHS/United StatesCA143231/CA/NCI NIH HHS/United StatesK99 CA125994/CA/NCI NIH HHS/United StatesK99 CA143231/CA/NCI NIH HHS/United StatesP50 GM107615/GM/NIGMS NIH HHS/United StatesR00 CA125994/CA/NCI NIH HHS/United StatesR00 CA143231/CA/NCI NIH HHS/United States2015-05-25T00:00:00Z26003204PMC449188

A computational framework to emulate the human perspective in flow cytometric data analysis

Background: In recent years, intense research efforts have focused on developing methods for automated flow cytometric data analysis. However, while designing such applications, little or no attention has been paid to the human perspective that is absolutely central to the manual gating process of identifying and characterizing cell populations. In particular, the assumption of many common techniques that cell populations could be modeled reliably with pre-specified distributions may not hold true in real-life samples, which can have populations of arbitrary shapes and considerable inter-sample variation. &lt;p/&gt;Results: To address this, we developed a new framework flowScape for emulating certain key aspects of the human perspective in analyzing flow data, which we implemented in multiple steps. First, flowScape begins with creating a mathematically rigorous map of the high-dimensional flow data landscape based on dense and sparse regions defined by relative concentrations of events around modes. In the second step, these modal clusters are connected with a global hierarchical structure. This representation allows flowScape to perform ridgeline analysis for both traversing the landscape and isolating cell populations at different levels of resolution. Finally, we extended manual gating with a new capacity for constructing templates that can identify target populations in terms of their relative parameters, as opposed to the more commonly used absolute or physical parameters. This allows flowScape to apply such templates in batch mode for detecting the corresponding populations in a flexible, sample-specific manner. We also demonstrated different applications of our framework to flow data analysis and show its superiority over other analytical methods. &lt;p/&gt;Conclusions: The human perspective, built on top of intuition and experience, is a very important component of flow cytometric data analysis. By emulating some of its approaches and extending these with automation and rigor, flowScape provides a flexible and robust framework for computational cytomics

Selective Uptake of Pelagic Microbial Community Members by Caribbean Reef Corals

Coral reefs are possible sinks for microbes; however, the removal mechanisms at play are not well understood. Here, we characterize pelagic microbial groups at the CARMABI reef (Curaçao) and examine microbial consumption by three coral species: Madracis mirabilis, Porites astreoides, and Stephanocoenia intersepta. Flow cytometry analyses of water samples collected from a depth of 10 m identified 6 microbial groups: Prochlorococcus, three groups of Synechococcus, photosynthetic eukaryotes, and heterotrophic bacteria. Minimum growth rates (µ) for Prochlorococcus, all Synechococcus groups, and photosynthetic eukaryotes were 0.55, 0.29, and 0.45µ day-1, respectively, and suggest relatively high rates of productivity despite low nutrient conditions on the reef. During a series of 5-h incubations with reef corals performed just after sunset or prior to sunrise, reductions in the abundance of photosynthetic picoeukaryotes, Prochlorococcus and Synechococcus cells, were observed. Of the three Synechococcus groups, one decreased significantly during incubations with each coral and the other two only with M. mirabilis. Removal of carbon from the water column is based on coral consumption rates of phytoplankton and averaged between 138 ng h-1and 387 ng h-1, depending on the coral species. A lack of coral-dependent reduction in heterotrophic bacteria, differences in Synechococcus reductions, and diurnal variation in reductions of Synechococcus and Prochlorococcus, coinciding with peak cell division, point to selective feeding by corals. Our study indicates that bentho-pelagic coupling via selective grazing of microbial groups influences carbon flow and supports heterogeneity of microbial communities overlying coral reefs. Importance We identify interactions between coral grazing behavior and the growth rates and cell abundances of pelagic microbial groups found surrounding a Caribbean reef. During incubation experiments with three reef corals, reductions in microbial cell abundance differed according to coral species and suggest specific coral or microbial mechanisms are at play. Peaks in removal rates of Prochlorococcus and Synechococcus cyanobacteria appear highest during postsunset incubations and coincide with microbial cell division. Grazing rates and effort vary across coral species and picoplankton groups, possibly influencing overall microbial composition and abundance over coral reefs. For reef corals, use of such a numerically abundant source of nutrition may be advantageous, especially under environmentally stressful conditions when symbioses with dinoflagellate algae break down

An expression tag toolbox for microbial production of membrane bound plant cytochromes P450

Membrane-associated Cytochromes P450 (P450s) are one of the most important enzyme families for biosynthesis of plant-derived medicinal compounds. However, the hydrophobic nature of P450s makes their use in robust cell factories a challenge. Here we explore a small library of N-terminal expression tag chimeras of the model plant P450 CYP79A1 in different Escherichia coli strains. Using a high-throughput screening platform based on C-terminal GFP fusions, we identify several highly expressing and robustly performing chimeric designs. Analysis of long-term cultures by flow cytometry showed homogeneous populations for some of the conditions. Three chimeric designs were chosen for a more complex combinatorial assembly of a multigene pathway consisting of two P450s and a redox partner. Cells expressing these recombinant enzymes catalysed the conversion of the substrate to highly different ratios of the intermediate and the final product of the pathway. Finally, the effect of a robustly performing expression tag was explored with a library of 49 different P450s from medicinal plants and nearly half of these were improved in expression by more than 2-fold. The developed toolbox serves as platform to tune P450 performance in microbial cells, thereby facilitating recombinant production of complex plant P450-derived biochemicals

Inflammation, caffeine and adenosine in neonatal hypoxic ischemic brain injury

Background: Brain injury during the neonatal period has potentially lifelong consequences for a child. Perinatal infections and inflammation can induce preterm birth and unfavorable cognitive development, Thus inflammation has received enthusiastic interest for potential therapeutic approaches seeking to protect the newborn brain. Experimental evidence demonstrates that inflammation induces brain injury succeeding the initial insult. A key cytokine in brain injury is the tumor necrosis factor (TNF-α), with reported detrimental cytotoxic effects on selected neuronal populations. Nonetheless, important functions of TNF-α in cerebral homeostasis and development have also been described. Caffeine is used against apneas of prematurity, with noticeable protection against cognitive delay and cerebral palsy. The main effects of caffeine at clinically relevant doses are mediated through inhibition of adenosine receptors. Adenosine is formed from adenosine triphosphate, the main transporter of chemical energy in the cell, which is readily cleaved to adenosine upon extracellular release or extensive leakage from injured necrotic cells. Hence, adenosine signaling is tightly interrelated with local energy levels and cell injury. In addition, adenosine modulates inflammatory responses in profound ways. Methods: In mouse models of premature excitotoxic lesions and full term hypoxic ischemic brain injury we investigated blockade of TNF-α with and without interleukin IL-1 or lipopolysaccharide (LPS) induced systemic inflammation. Furthermore, in the hypoxic ischemic model we developed a flow cytometry based method to investigate temporal distribution of brain infiltrating and splenetic immune cells and their activation. To analyze the data in an unbiased way, we next adapted a data driven gating methodology. Moreover, we used principal component analysis to discriminate between experimentally entangled variables. Utilizing these techniques, we explored the effect of genetic inactivation of adenosine A1 and A2A receptors in the hypoxic ischemic model. We also tested the unselective, competitive adenosine receptor antagonist caffeine and assessed the effect on outcome and immune activation. Results: Blockade of TNF-α protected the brain against excitotoxic lesions in the presence but not absence of systemic inflammation. No protection was observed in the full term hypoxic ischemic model. Persistent lymphocyte activation was found three months after the lesion. Moreover, spleenocytes harvested five months after neonatal brain damage proliferated when stimulated with brain homogenate in contrast to sham operated counterparts. Adenosine A1 receptor deficient mice acquired significantly larger infarcts and associated adverse behavioral outcome compared to wild type. There were specific alterations in the immune responses induced after brain injury, including impaired cytotoxic function and dysregulation of regulatory B-lymphocytes. Adenosine A2A receptor knockout mice developed increased atrophy compared to wild type after hypoxic ischemia, an effect accompanied by functional deficits in behavioral tests. Furthermore, a compensated functional insufficiency was estimated in the regulatory T- lymphocyte compartment in combination with a seemingly inadequate number of myeloid derived suppressor like cells, accompanied by a reversed, increased response in innate antigen presenting cells in the knockout. Finally, we report neuroprotective properties of 5 mg/kg caffeine given directly after neonatal brain injury. Discussion: TNF-α blockade could potentially protect against preterm excitotoxic brain injury. Only patients with concurrent systemic inflammation would potentially benefit. Moreover, concern about adverse effects exists, why TNF-α blockade for neonatal brain injury is likely not clinically applicable in the near future. Persistent long term cerebral adaptive immune activation, preceded by systemic immune activation in spleen was discovered. Remarkably, spleenocytes from animals subjected to brain injury responded to brain antigen five months after brain damage, whereas spleenocytes from uninjured did not, suggesting formation of immunological memory that might affect long term outcome and provoke autoimmunity later in life. To avoid bias from manual gating of flow cytometry data we developed a data driven approach adapted for brain infiltrating immune cells. Furthermore, we deployed principal component analysis to verify biological relevance in the pattern of immune activation and to discriminate between genotype and injury size effects, since they are experimentally inseparable. Thus we could predict genotype and whether they acquired brain injury or not, from the flow cytometric immune activation pattern alone. Adenosine A1 receptor deficient mice display signs of regulatory B-lymphocyte dysfunction that imply a novel adenosinergic mechanism of B-lymphocyte regulation. In addition, these animals displayed signs of altered cellular cytotoxic immunity. Thus considerable effects on immune activation were present in the A1 receptor knockouts compared to wild type, adding another mechanism linked to worse outcome after hypoxic ischemic brain injury in these animals. Deletion of the adenosine A2A receptor similarly causes worse outcome, however, the alteration of the immune response is completely different. Fundamental changes were observed in regulatory populations like monocyte derived suppressor like cells and regulatory T-lymphocytes. Extensive activation of cytotoxic populations in the adenosine A2A receptor knockout links insufficient regulatory immune function with adverse behavioral and morphological outcome. We also propose a novel hypothesis that short term blockade of adenosine A2A receptors offers neuroprotection whereas long term blockade is detrimental by immunological mechanisms. Thus we tested the tentative therapeutic potential of caffeine, an unselective competitive antagonist of adenosine receptors. Caffeine 5mg/kg given directly after the insult resulted in reduced injury size after neonatal hypoxic ischemia. Since caffeine is a relatively well studied substance with negligible adverse long term effect in technically sound studies absent of significant bias, this approach has a clear clinical relevance. Are we ready for a clinical trial