My winding career path.

A look at possible career options open to academic researchers, both inside and outside universities.

A Flow Cytometry Method for Rapidly Assessing Mycobacterium tuberculosis Responses to Antibiotics with Different Modes of Action.

Current methods for assessing the drug susceptibility of Mycobacterium tuberculosis are lengthy and do not capture information about viable organisms that are not immediately culturable under standard laboratory conditions as a result of antibiotic exposure. We have developed a rapid dual-fluorescence flow cytometry method using markers for cell viability and death. We show that the fluorescent marker calcein violet with an acetoxy-methyl ester group (CV-AM) can differentiate between populations of M. tuberculosis growing at different rates, while Sytox green (SG) can differentiate between live and dead mycobacteria. M. tuberculosis was exposed to isoniazid or rifampin at different concentrations over time and either dual stained with CV-AM and SG and analyzed by flow cytometry or plated to determine the viability of the cells. Although similar trends in the loss of viability were observed when the results of flow cytometry and the plate counting methods were compared, there was a lack of correlation between these two approaches, as the flow cytometry analysis potentially captured information about cell populations that were unable to grow under standard conditions. The flow cytometry approach had an additional advantage in that it could provide insights into the mode of action of the drug: antibiotics targeting the cell wall gave a flow cytometry profile distinct from those inhibiting intracellular processes. This rapid drug susceptibility testing method could identify more effective antimycobacterials, provide information about their potential mode of action, and accelerate their progress to the clinic.

Detection of cytoplasmic nucleophosmin expression by imaging flow cytometry.

Mutations within the nucleophosmin NPM1 gene occur in approximately one-third of cases of acute myeloid leukemia (AML). These mutations result in cytoplasmic accumulation of the mutant NPM protein. NPM1 mutations are currently detected by molecular methods. Using samples from 37 AML patients, we investigated whether imaging flow cytometry could be a viable alternative to this current technique. Bone marrow/peripheral blood cells were stained with anti-NPM antibody and DRAQ5 nuclear stain, and data were acquired on an ImageStream imaging flow cytometer (Amnis Corp., Seattle, USA). Using the similarity feature for data analysis, we demonstrated that this technique could successfully identify cases of AML with a NPM1 mutation based on cytoplasmic NPM protein staining (at similarity threshold of 1.1 sensitivity 88% and specificity 90%). Combining data of mean fluorescence intensity and % dissimilar staining in a 0-2 scoring system further improved the sensitivity (100%). Imaging flow cytometry has the potential to be included as part of a standard flow cytometry antibody panel to identify potential NPM1 mutations as part of diagnosis and minimal residual disease monitoring. Imaging flow cytometry is an exciting technology that has many possible applications in the diagnosis of hematological malignancies, including the potential to integrate modalities.

Multispectral imaging flow cytometry reveals distinct frequencies of γ-H2AX foci induction in DNA double strand break repair defective human cell lines.

The measurement of γ-H2AX foci induction in cells provides a sensitive and reliable method for the quantitation of DNA damage responses in a variety of cell types. Accurate and rapid methods to conduct such observations are desirable. In this study, we have employed the novel technique of multispectral imaging flow cytometry to compare the induction and repair of γ-H2AX foci in three human cell types with different capacities for the repair of DNA double strand breaks (DSB). A repair normal fibroblast cell line MRC5-SV1, a DSB repair defective ataxia telangiectasia (AT5BIVA) cell line, and a DNA-PKcs deficient cell line XP14BRneo17 were exposed to 2 Gy gamma radiation from a (60)Cobalt source. Thirty minutes following exposure, we observed a dramatic induction of foci in the nuclei of these cells. After 24 hrs, there was a predictable reduction on the number of foci in the MRC5-SV1 cells, consistent with the repair of DNA DSB. In the AT5BIVA cells, persistence of the foci over a 24-hr period was due to the failure in the repair of DNA DSB. However, in the DNA-PKcs defective cells (XP14BRneo17), we observed an intermediate retention of foci in the nuclei indicative of partial repair of DNA DSB. In summary, the application of imaging flow cytometry has permitted an evaluation of foci in a large number of cells (20,000) for each cell line at each time point. This provides a novel method to determine differences in repair kinetics between different cell types. We propose that imaging flow cytometry provides an alternative platform for accurate automated high through-put analysis of foci induction in a variety of cell types.

Recombinant VP4 of human rhinovirus induces permeability in model membranes.

In common with all nonenveloped viruses, the mechanism of picornavirus membrane penetration during cell entry is poorly understood. The small, myristylated capsid protein VP4 has been implicated in this process. Here we show that recombinant VP4 of human rhinovirus 16 has the ability to associate with and induce membrane permeability in otherwise intact liposomes. This provides further evidence that VP4 plays a key role in picornavirus cell entry.

A flow cytometric assay for analysis of natural-killer cell-mediated cytolysis of adenovirus-transformed cells.

Natural-killer (NK) cells play an important role in recognizing and eliminating virally infected and transformed cells. To study this process, convenient assays for NK-cell function are required. Conventional NK-cell activity assays measure the release of 51Cr from prelabeled target cells following membrane disruption. This chapter describes nonradiometric assays for NK-cell killing of adenovirus-transformed human cells that can be applied to multiple cell samples using flow cytometry.

Flow cytometric detection of adenoviruses and intracellular adenovirus proteins.

Precise and simple assay of purified and crude preparations of human adenoviruses is essential for basic and gene therapy research. Previous bioassays used to quantitate adenoviruses (such as the plaque assay or fluorescent focus assay) are time-consuming and subjective in their interpretation. Here we describe a flow cytometric method that eliminates these disadvantages and provides a quantitative and reliable method of focus-forming unit (FFU) assay.

Differential expression of LFA-3, Fas and MHC Class I on Ad5- and Ad12-transformed human cells and their susceptibility to lymphokine-activated killer (LAK) cells.

Adenovirus (Ad) E1A is a potent oncogene and has been shown to deregulate the expression of a large number of cellular genes leading to cellular transformation. Here we have analysed the expression of several immunomodulatory molecules on the surface of a set of human cell lines transformed with either Ad12 or Ad5. Human cells transformed with Ad12 demonstrated reduced expression of cell surface LFA-3, Fas and MHC class I when compared to Ad5-transformed cells. Furthermore, Ad12-transformed human cell lines demonstrated greater susceptibility to lysis by lymphokine-activated killer (LAK) cells, compared to Ad5-transformed human cell lines. In contrast, previous studies with rodent cells showed that both Ad5- and Ad12-transformed rat cells were susceptible to LAK cells. Thus, transformation of human cells with Ad5 or Ad12 results in differences in the expression of immunomodulatory molecules on the cell surface and differential recognition of these virus-transformed cells by immune effector cells.