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.

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 60Cobalt 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. © 2011 International Society for Advancement of Cytometry

High-risk human papillomavirus (HPV) is a major causative agent of cervical cancer and the E6 and E7 genes encode the major HPV oncoproteins. The E7 protein from high-risk HPV types alters cell cycle progression and represses genes encoding components of the antigenpresentation pathway, suggesting a role for E7 in tumour immune evasion. We show that knockdown of E7 expression in HPV16-and HPV18-transformed cervical carcinoma cells by RNA interference increased expression of major histocompatibility complex (MHC) class I at the cell surface and reduced susceptibility of these cells to natural killer (NK) cells. Tetracycline-regulated induction of HPV16 E7 resulted in reduced expression of cell surface MHC class I molecules and increased NK cell killing. Our results suggest that, for HPV-associated malignancies, reduced MHC class I expression is the result of an active immune evasion strategy that has evolved to assist viral replication. The high-risk human papillomavirus (HPV) types 16 (HPV16) and 18 (HPV18) have a strong association with the incidence of cervical cancer, with at least 90% of cervical adenocarcinomas harbouring HPV16 or HPV18 DNA (Walboomers et al., 1999). The E6 and E7 oncogenes are continuously expressed following cellular transformation and in cervical cancer tissues while expression of the E5 gene contributes to transformation but is not expressed in cervical tumours (Howley and Lowy, 2007). Many viruses and tumours evade the T-cell-mediated immune response, primarily by reducing the levels of surface MHC class I, thus reducing the presentation of viral/tumour antigens (Garcia-Lora et al., 2003;Hewitt, 2003). We have previously shown that the E7 oncoproteins of the high-risk HPV16 and 18 types are able to repress the activity of promoters encoding components of the MHC class I antigen processing and presentation pathway (Georgopoulos et al., 2000) and recent data further suggest that HPV16 E7 interacts with the MHC class I heavy chain promoter in HPV16-transformed human cells (Li et al., 2006). Here, we have used two complementary approaches to evaluate the effect of HPV16 and 18 E7 expression on the levels of cell-surface MHC class I molecules and consequent effects on natural killer (NK) cell recognition.In the first approach, E7 expression was reduced in HPV-transformed cells by transfection of an E7-specific short interfering RNA (siRNA) molecule (Jiang and Milner, 2002;Hall and Alexander, 2003). HPV16-transformed cells (SiHa and CaSki) and HPV18-transformed cells (HeLa) were mock-transfected or transfected with E7-specific or control siRNAs and the level of E7 mRNA analysed 72 h post-transfection by quantitative real-time RT-PCR (Figure 1a). This showed that the E7-specific siRNA reduced E7 mRNA sequences by approximately 90% in the HPV16-and HPV18-transformed cell lines. Treatment of the three HPV-transformed cell lines with E7-specific siRNA induced growth arrest and profound morphological changes in transfected cells, as described previously (Hall and Alexander, 2003); however, these ...

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.

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. ' 2012 International Society for Advancement of Cytometry Key termsimaging flow cytometry; acute leukemia; nucleophosmin; hematology NUCLEOPHOSMIN (NPM) is both a nuclear (predominantly nucleolar) and cytoplasmic protein that functions as a molecular chaperone (1). It has many functions including the prevention of protein aggregation in the nucleolus and regulation of p53 levels (2). Mutations within the C-terminal region (exon 12) of the NPM1 gene occur in approximately one-third of cases of de novo acute myeloid leukemia (AML) (3,4) leading to increased nuclear export and aberrant cytoplasmic accumulation of the mutant NPM protein (5). A number of methods have been assessed to detect cytoplasmic NPM (cNPM) in clinical specimens. Immunohistochemistry has been reported to be reliable, especially on B5-fixed bone marrow trephines, with close correlation between cNPM and the presence of mutated NPM1 (6). Immunocytochemistry of air-dried blood and bone marrow smears can also detect cNPM in myeloblasts but is insufficiently reliable for diagnostic use (7). Fluorescent confocal microscopy and standard flow cytometry have both been evaluated for cNPM detection (8,9). Standard flow cytometry cannot be used to assess the localization of molecules within specific cellular compartments, but two alternative approaches have been assessed. The first, using antibodies to mutant NPM1, was able to identify NPM1-mutated AML cases exclusively with or without the addition of a de novo NPM ''control'' antibody (8,9). The second method specifically addressed mean fluorescence intensity (MFI) and showed higher MFI in NPM1 mutated than wild-type AML cases (10). However, discrepancie...