Gérald Grégori

Resolution of viable and membrane-compromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining.

ABSTRACT The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214–218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.

Sustained activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase pathways by hepatitis B virus X protein mediates apoptosis via induction of Fas/FasL and tumor necrosis factor (TNF) receptor 1/TNF-α expression

ABSTRACT Activation of the cellular stress pathways (c-Jun N-terminal kinase [JNK] and p38 mitogen-activated protein [MAP] kinase) is linked to apoptosis. However, whether both pathways are required for apoptosis remains unresolved. Hepatitis B virus X protein (pX) activates p38 MAP kinase and JNK pathways and, in response to weak apoptotic signals, sensitizes hepatocytes to apoptosis. Employing hepatocyte cell lines expressing pX, which was regulated by tetracycline, we investigated the mechanism of apoptosis by p38 MAP kinase and JNK pathway activation. Inhibition of the p38 MAP kinase pathway rescues by 80% the initiation of pX-mediated apoptosis, whereas subsequent apoptotic events involve both pathways. pX-mediated activation of p38 MAP kinase and JNK pathways is sustained, inducing the transcription of the death receptor family genes encoding Fas/FasL and tumor necrosis factor receptor 1 (TNFR1)/TNF-α and the p53-regulated Bax and Noxa genes. The pX-dependent expression of Fas/FasL and TNFR1/TNF-α mediates caspase 8 activation, resulting in Bid cleavage. In turn, activated Bid, acting with pX-induced Bax and Noxa, mediates the mitochondrial release of cytochrome c, resulting in the activation of caspase 9 and apoptosis. Combined antibody neutralization of FasL and TNF-α reduces by 70% the initiation of pX-mediated apoptosis. These results support the importance of the pX-dependent activation of both the p38 MAP kinase and JNK pathways in pX-mediated apoptosis and suggest that this mechanism of apoptosis occurs in vivo in response to weak apoptotic signals.

Hepatitis B virus X protein increases the Cdt1-to-geminin ratio inducing DNA re-replication and polyploidy.

Hepatitis B virus X protein (pX) is implicated in hepatocellular carcinoma pathogenesis by an unknown mechanism. Employing the tetracycline-regulated pX-expressing 4pX-1 cell line, derived from the murine AML12 hepatocyte cell line, we demonstrate that pX induces partial polyploidy (>4N DNA). Depletion of p53 in 4pX-1 cells increases by 5-fold the polyploid cells in response to pX expression, indicating that p53 antagonizes pX-induced polyploidy. Dual-parameter flow cytometric analyses show pX-dependent bromodeoxyuridine (BrdUrd) incorporation in 4pX-1 cells containing 4N and >4N DNA, suggesting pX induces DNA re-replication. Interestingly, pX increases expression of endogenous replication initiation factors Cdc6 and Cdtl while suppressing geminin expression, a negative regulator of rereplication. In comparison to a geminin knockdown 4pX-1 cell line used as DNA re-replication control, the Cdt1/geminin ratio is greater in 4pX-1 cells expressing pX, indicating that pX promotes DNA re-replication. In support of this conclusion, pX-expressing 4pX-1 cells, similar to the geminin knockdown 4pX-1 cells, continue to incorporate BrdUrd in the G2 phase and exhibit nuclear Cdc6 and MCM5 co-localization and the absence of geminin. In addition, pX expression activates the ATR kinase, the sensor of DNA re-replication, which in turn phosphorylates RAD17 and H2AX. Interestingly, phospho-H2AX-positive and BrdUrd -positive cells progress through mitosis, demonstrating a link between pX-induced DNA re-replication and polyploidy. Our studies high-light a novel function of pX that likely contributes to hepatocellular carcinoma pathogenesis.

Hyperspectral cytometry at the single-cell level using a 32-channel photodetector

Despite recent progress in cell‐analysis technology, rapid classification of cells remains a very difficult task. Among the techniques available, flow cytometry (FCM) is considered especially powerful, because it is able to perform multiparametric analyses of single biological particles at a high flow rate–up to several thousand particles per second. Moreover, FCM is nondestructive, and flow cytometric analysis can be performed on live cells. The current limit for simultaneously detectable fluorescence signals in FCM is around 8–15 depending upon the instrument. Obtaining multiparametric measurements is a very complex task, and the necessity for fluorescence spectral overlap compensation creates a number of additional difficulties to solve. Further, to obtain well‐separated single spectral bands a very complex set of optical filters is required. This study describes the key components and principles involved in building a next‐generation flow cytometer based on a 32‐channel PMT array detector, a phase‐volume holographic grating, and a fast electronic board. The system is capable of full‐spectral data collection and spectral analysis at the single‐cell level. As demonstrated using fluorescent microspheres and lymphocytes labeled with a cocktail of antibodies (CD45/FITC, CD4/PE, CD8/ECD, and CD3/Cy5), the presented technology is able to simultaneously collect 32 narrow bands of fluorescence from single particles flowing across the laser beam in <5 μs. These 32 discrete values provide a proxy of the full fluorescence emission spectrum for each single particle (cell). Advanced statistical analysis has then been performed to separate the various clusters of lymphocytes. The average spectrum computed for each cluster has been used to characterize the corresponding combination of antibodies, and thus identify the various lymphocytes subsets. The powerful data‐collection capabilities of this flow cytometer open up significant opportunities for advanced analytical approaches, including spectral unmixing and unsupervised or supervised classification.

Phytoplankton group dynamics in the Bay of Marseilles during a 2-year survey based on analytical flow cytometry

BACKGROUND The Bay of Marseilles is under the influence of a large urban concentration and its maritime activities. All of them discharge compounds (hydrocarbons, excess nutrients, heavy metals, chemicals, etc.) that can alter the marine ecosystem. To investigate whether ultraphytoplankton (<10 microm) could be used as biosensors for their own ecosystem, a 2-year survey was conducted in the Bay of Marseilles. METHODS Seven stations monitored water mass and potential anthropic effects in the bay. Seawater samples were collected monthly or bimonthly at three depths, prefiltered, fixed, and kept in liquid nitrogen until flow cytometric analysis. RESULTS Five categories were created: Prochlorococcus, picoeukaryotes (<2 microm), nanoeukaryotes I (2--6 microm), nanoeukaryotes II (6--10 microm), and Synechococcus (<1.5 microm). Artificial neural network analysis (Kohonen self-organizing maps) produced the same number of clusters as cluster analysis with Winlist software (Verity Software House). CONCLUSIONS In addition to the wide variabilities in abundance and biomass, there were a strong seasonal signal and sporadic events. Lessons are derived from this study for future monitoring of marine microorganisms.

Integrating Cytomics and Proteomics

Systems biology along with what is now classified as cytomics provides an excellent opportunity for cytometry to become integrated into studies where identification of functional proteins in complex cellular mixtures is desired. The combination of cell sorting with rapid protein-profiling platforms offers an automated and rapid technique for greater clarity, accuracy, and efficiency in identification of protein expression differences in mixed cell populations. The integration of cell sorting to purify cell populations opens up a new area for proteomic analysis. This article outlines an approach in which well defined cell analysis and separation tools are integrated into the proteomic programs within a core laboratory. In addition we introduce the concepts of flow cytometry sorting to demonstrate the importance of being able to use flow cytometry as a cell separation technology to identify and collect purified cell populations. Data demonstrating the speed and versatility of this combination of flow cytometry-based cell separation and protein separation and subsequent analysis, examples of protein maps from purified sorted cells, and an analysis of the overall procedure will be shown. It is clear that the power of cell sorting to separate heterogeneous populations of cells using specific phenotypic characteristics increases the power of rapid automated protein separation technologies.

Antifouling Coatings Influence both Abundance and Community Structure of Colonizing Biofilms: a Case Study in the Northwestern Mediterranean Sea

ABSTRACT When immersed in seawater, substrates are rapidly colonized by both micro- and macroorganisms. This process is responsible for important economic and ecological prejudices, particularly when related to ship hulls or aquaculture nets. Commercial antifouling coatings are supposed to reduce biofouling, i.e., micro- and macrofoulers. In this study, biofilms that primarily settled on seven different coatings (polyvinyl chloride [PVC], a fouling release coating [FRC], and five self-polishing copolymer coatings [SPC], including four commercial ones) were quantitatively studied, after 1 month of immersion in summer in the Toulon Bay (Northwestern Mediterranean Sea, France), by using flow cytometry (FCM), microscopy, and denaturing gradient gel electrophoresis. FCM was used after a pretreatment to separate cells from the biofilm matrix, in order to determine densities of heterotrophic bacteria, picocyanobacteria, and pico- and nanoeukaryotes on these coatings. Among diatoms, the only microphytobenthic class identified by microscopy, Licmophora, Navicula, and Nitzschia were determined to be the dominant taxa. Overall, biocide-free coatings showed higher densities than all other coatings, except for one biocidal coating, whatever the group of microorganisms. Heterotrophic bacteria always showed the highest densities, and diatoms showed the lowest, but the relative abundances of these groups varied depending on the coating. In particular, the copper-free SPC failed to prevent diatom settlement, whereas the pyrithione-free SPC exhibited high picocyanobacterial density. These results highlight the interest in FCM for antifouling coating assessment as well as specific selection among microbial communities by antifouling coatings.

Generalized unmixing model for multispectral flow cytometry utilizing nonsquare compensation matrices

Multispectral and hyperspectral flow cytometry (FC) instruments allow measurement of fluorescence or Raman spectra from single cells in flow. As with conventional FC, spectral overlap results in the measured signal in any given detector being a mixture of signals from multiple labels present in the analyzed cells. In contrast to traditional polychromatic FC, these devices utilize a number of detectors (or channels in multispectral detector arrays) that is larger than the number of labels, and no particular detector is a priori dedicated to the measurement of any particular label. This data‐acquisition modality requires a rigorous study and understanding of signal formation as well as unmixing procedures that are employed to estimate labels abundance. The simplest extension of the traditional compensation procedure to multispectral data sets is equivalent to an ordinary least‐square (LS) solution for estimating abundance of labels in individual cells. This process is identical to the technique employed for unmixing spectral data in various imaging fields. The present study shows that multispectral FC data violate key assumptions of the LS process, and use of the LS method may lead to unmixing artifacts, such as population distortion (spreading) and the presence of negative values in biomarker abundances. Various alternative unmixing techniques were investigated, including relative‐error minimization and variance‐stabilization transformations. The most promising results were obtained by performing unmixing using Poisson regression with an identity‐link function within a generalized linear model framework. This formulation accounts for the presence of Poisson noise in the model of signal formation and subsequently leads to superior unmixing results, particularly for dim fluorescent populations. The proposed Poisson unmixing technique is demonstrated using simulated 8‐channel, 2‐fluorochrome data and real 32‐channel, 6‐fluorochrome data. The quality of unmixing is assessed by computing absolute and relative errors, as well as by calculating the symmetrized Kullback–Leibler divergence between known and approximated populations. These results are applicable to any flow‐based system with more detectors than labels where Poisson noise is the dominant contributor to the overall system noise and highlight the fact that explicit incorporation of appropriate noise models is the key to accurately estimating the true label abundance on the cells.

Functional analysis and classification of phytoplankton based on data from an automated flow cytometer

Analytical flow cytometry (FCM) is well suited for the analysis of phytoplankton communities in fresh and sea waters. The measurement of light scatter and autofluorescence properties of particles by FCM provides optical fingerprints, which enables different phytoplankton groups to be separated. A submersible version of the CytoSense flow cytometer (the CytoSub) has been designed for in situ autonomous sampling and analysis, making it possible to monitor phytoplankton at a short temporal scale and obtain accurate information about its dynamics. For data analysis, a manual clustering is usually performed a posteriori: data are displayed on histograms and scatterplots, and group discrimination is made by drawing and combining regions (gating). The purpose of this study is to provide greater objectivity in the data analysis by applying a nonmanual and consistent method to automatically discriminate clusters of particles. In other words, we seek for partitioning methods based on the optical fingerprints of each particle. As the CytoSense is able to record the full pulse shape for each variable, it quickly generates a large and complex dataset to analyze. The shape, length, and area of each curve were chosen as descriptors for the analysis. To test the developed method, numerical experiments were performed on simulated curves. Then, the method was applied and validated on phytoplankton cultures data. Promising results have been obtained with a mixture of various species whose optical fingerprints overlapped considerably and could not be accurately separated using manual gating.

Stochastic activation of the response regulator PhoB by noncognate histidine kinases

Abstract Two-component systems (TCS) are the most prevalent gene regulatory mechanism in bacteria. A typical TCS is comprised of a histidine kinase (HK) and a partner response regulator (RR). Specific environment signals lead to autophosphorylation of different HKs, which in turn act as phosphoryl donors for autophosphorylation of their partner RRs. Nonpartner HKs and RRs also interact, giving rise to cross regulation among TCSs in response to diverse signals. PhoR (HK) and PhoB (RR) constitute the TCS for detection of environmental (extracellular) inorganic phosphate (Pi). The PhoR/PhoB TCS controls the expression of a large number of genes for acquisition of alternative phosphorus sources, including phoA, which encodes the non-specific phosphohydrolase bacterial alkaline phosphatase (Bap). Cross activation of PhoB by the nonpartner HK CreC is now a classic example of cross regulation among TCSs. A systematic search for other cross talking HKs revealed five additional HKs that activate (phosphorylate) PhoB (J. M. B. and B. L. W., unpublished data). Examination of cross activation of PhoB by these non-partner HKs by flow cytometry at the single-cell level revealed a bimodal, “all-or-none,” distribution pattern for expression of a phoAp-gfp (green fluorescent protein) reporter fusion. Although the basis of the observed stochastic behavior is unclear, it seems to reflect an inherent property of TCSs. We propose that cells exploit the stochastic character of TCSs to achieve nongenetic (epigenetic) diversity within genetically homogeneous cell populations in order to facilitate adaptation to environmental changes.