Detection of virulence genes in Malaysian Shigella species by multiplex PCR assay

BACKGROUND: In Malaysia, Shigella spp. was reported to be the third commonest bacterial agent responsible for childhood diarrhoea. Currently, isolation of the bacterium and confirmation of the disease by microbiological and biochemical methods remain as the "gold standard". This study aimed to detect the prevalence of four Shigella virulence genes present concurrently, in randomly selected Malaysian strains via a rapid multiplex PCR (mPCR) assay. METHODS: A mPCR assay was designed for the simultaneous detection of chromosomal- and plasmid-encoded virulence genes (set1A, set1B, ial and ipaH) in Shigella spp. One hundred and ten Malaysian strains (1997–2000) isolated from patients from various government hospitals were used. Reproducibility and sensitivity of the assay were also evaluated. Applicability of the mPCR in clinical settings was tested with spiked faeces following preincubation in brain heart infusion (BHI) broth. RESULTS: The ipaH sequence was present in all the strains, while each of the set1A, set1B and ial gene was present in 40% of the strains tested. Reproducibility of the mPCR assay was 100% and none of the non-Shigella pathogens tested in this study were amplified. The mPCR could detect 100 colony-forming units (cfu) of shigellae per reaction mixture in spiked faeces following preincubation. CONCLUSIONS: The mPCR system is reproducible, sensitive and is able to identify pathogenic strains of shigellae irrespective of the locality of the virulence genes. It can be easily performed with a high throughput to give a presumptive identification of the causal pathogen

Characterization of Tumourigenic subpopulations in Nasopharyngeal carcinoma isolated by side population and cancer stem cell marker approaches / Susan Hoe Ling Ling

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy of the nasopharynx. Studies on NPC tumourigenic subpopulations are often performed in NPC cell lines due to a lack of sizeable NPC tissues. Commonly used methods to identify the subpopulations include sphere-forming culture, side population (SP) assay and cancer stem cell (CSC) markers. The main objective of this study is to characterize the biological properties of tumourigenic cells in NPC isolated separately using SP assay and CSC markers from established NPC cell lines and/or early-passage NPC patient-derived xenografts (PDXs). SP assay identifies stem-like cells by their ability to extrude Hoechst 33342 dye. Using this first approach, SP cells were identified in HK1, a recurrent NPC cell line and in xeno-284, an early-passage NPC PDX established from recurrent metastatic NPC. HK1 contained 5 to 10% of SP cells while xeno-284 had less than 0.5% SP cells. HK1 SP cells significantly formed more holoclones than its non-SP (NSP) cells, an in vitro clone morphology closely related to self-renewal. SP cells also had higher aldehyde dehydrogenase (ALDH) activity than NSP cells, showed asymmetrical cell division and contained slow-proliferating cells. ABCG2, SOX2, TERT, MYC as well as certain Hedgehog, Notch, TGF- and Wnt signalling pathway transcripts were significantly upregulated in SP cells. Despite significant differences seen in vitro and in gene expression experiments, SP and NSP cells showed an overall comparable tumour formation ability and tumour-initiating cell (TIC) frequency in nude mice. CSC markers are successfully used to identify tumourigenic stem-like cells in solid tumours. In the second approach, CD24, CD44, EpCAM and a combination of EpCAM/CD44 markers were used to isolate subpopulations of cells from C666-1, a cell line established from an undifferentiated NPC biopsy and from xeno-B110, an early- passage NPC PDX established from a NPC biopsy. CD44br and EpCAMbr cells from C666-1 and xeno-B110 enriched for faster-growing tumourigenic cells with resulting larger tumours. Marked growth differences in xeno-B110 were associated with higher percentage of S-phase cells and mitotic figures in the marker bright groups as compared to marker dim groups. EpCAM/CD44dbr marker from xeno-B110 did not enhance for faster-growing cells or higher TIC frequency than CD44br marker alone. CD24br, CD44br and EpCAMbr cells from xeno-B110 were more enriched for TICs than their respective dim phenotypes in the first passage and retained self-renewal property upon serial transplantation in vivo for three successive passages. At the final passage of serial transplantation, CD24br cells were 10.55 folds more enriched with TICs than CD24dim cells, CD44br cells had a 7.07-fold enrichment over CD44dim cells and there was a 4.89-fold TIC enrichment for EpCAMbr cells over EpCAMdim cells. KLF4 and CDKN1A transcripts were downregulated in all bright phenotypes which also had induced larger tumour growth, indicating a tumour suppressor role for KLF4 in NPC. Together, the study has shown that SP and CSC markers could isolate NPC cells with differential biological properties in vitro

Analysis of NK-92 cytotoxicity in nasopharyngeal carcinoma cell lines and patient-derived xenografts using impedance-based growth method

Natural killer (NK) cells are innate immune cells that can remove viral-infected tumour cells without antigen priming. This characteristic offers NK cells an edge over other immune cells as a potential therapy for nasopharyngeal carcinoma (NPC). In this study, we report how cytotoxicity was evaluated in target NPC cell lines and patient-derived xenograft (PDX) cells with effector NK-92, a commercially available NK cell line, by using xCELLigence RTCA system (a real-time, label-free impedance-based monitoring platform). Cell viability, proliferation and cytotoxicity were examined by RTCA. Cell morphology, growth and cytotoxicity were also monitored by microscopy. RTCA and microscopy showed that both target and effector cells were able to proliferate normally and to maintain original morphology in co-culture medium as they were in their own respective culture medium. As target and effector (T:E) cell ratios increased, cell viability as measured by arbitrary cell index (CI) values in RTCA decreased in all cell lines and PDX cells. NPC PDX cells were more sensitive to the cytotoxicity effect of NK-92 cells, than the NPC cell lines. These data were substantiated by GFP-based microscopy. We have shown how the RTCA system can be used for a high throughput screening of the effects of NK cells in cancer studies to obtain data such as cell viability, proliferation and cytotoxicity

Dynamic tracking of human umbilical cord mesenchymal stem cells (hUC-MSCs) following intravenous administration in mice model

Introduction: In the past decades, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have sparked interest in cellular therapy due to their immunomodulatory properties. Nevertheless, the fate of hUC-MSCs in the body remains poorly understood. This study aimed to investigate the biodistribution, homing and clearance of systemically administered hUC-MSCs in healthy BALB/c mice model. Methods: hUC-MSCs were labelled with GFP-Luc2 protein, followed by characterisation with flow cytometry. Upon intravenous infusion of transduced hUC-MSCs into the healthy BALB/c mice, the cells were dynamically monitored through the bioluminescent imaging (BLI) approach. Results: Transduction of hUC-MSCs with GFP-Luc2 not only preserved the characteristics of MSCs, but also allowed live monitoring of transduced cells in the mice model. Upon systemic administration, BLI showed that transduced hUC-MSCs first localised predominantly in the lungs of healthy BALB/c mice and mainly remained in the lungs for up to 3 days before eventually cleared from the body. At terminal sacrifice, plasma chemistry biomarkers remained unchanged except for C-peptide levels, which were significantly reduced in the hUC-MSCs group. Histopathological findings further revealed that hUC-MSCs infusion did not cause any adverse effects and toxicity to lung, liver and heart tissues. Conclusions: Collectively, systemically administrated hUC-MSCs was safe and demonstrated dynamic homing capacity before eventually disappearing from the body

Dielectrophoretic deformation of breast cancer cells for lab on a chip applications

This paper presents the development and experimental analysis of a curved microelectrode platform for the DEP deformation of breast cancer cells (MDA-MB-231). The platform is composed of arrays of curved DEP microelectrodes which are patterned onto a glass slide and samples containing MDA-MB-231 cells are pipetted onto the platform's surface. Finite element method is utilised to characterise the electric field gradient and DEP field. The performance of the system is assessed with MDA-MB-231 cells in a low conductivity 1% DMEM suspending medium. We applied sinusoidal wave AC potential at peak to peak voltages of 2, 5, and 10 Vpp at both 10 kHz and 50 MHz. We observed cell blebbing and cell shrinkage and analyzed the percentage of shrinkage of the cells. The experiments demonstrated higher percentage of cell shrinkage when cells are exposed to higher frequency and peak to peak voltage electric field.</p

Dielectrophoretic deformation of breast cancer cells for lab on a chip applications

This paper presents the development and experimental analysis of a curved microelectrode platform for the DEP deformation of breast cancer cells (MDA-MB-231). The platform is composed of arrays of curved DEP microelectrodes which are patterned onto a glass slide and samples containing MDA-MB-231 cells are pipetted onto the platform's surface. Finite element method is utilised to characterise the electric field gradient and DEP field. The performance of the system is assessed with MDA-MB-231 cells in a low conductivity 1% DMEM suspending medium. We applied sinusoidal wave AC potential at peak to peak voltages of 2, 5, and 10 Vpp at both 10 kHz and 50 MHz. We observed cell blebbing and cell shrinkage and analyzed the percentage of shrinkage of the cells. The experiments demonstrated higher percentage of cell shrinkage when cells are exposed to higher frequency and peak to peak voltage electric field

Dielectrophoresis-based microfluidic platforms for cancer diagnostics

The recent advancement of dielectrophoresis (DEP)-enabled microfluidic platforms is opening new opportunities for potential use in cancer disease diagnostics. DEP is advantageous because of its specificity, low cost, small sample volume requirement, and tuneable property for microfluidic platforms. These intrinsic advantages have made it especially suitable for developing microfluidic cancer diagnostic platforms. This review focuses on a comprehensive analysis of the recent developments of DEP enabled microfluidic platforms sorted according to the target cancer cell. Each study is critically analyzed, and the features of each platform, the performance, added functionality for clinical use, and the types of samples, used are discussed. We address the novelty of the techniques, strategies, and design configuration used in improving on existing technologies or previous studies. A summary of comparing the developmental extent of each study is made, and we conclude with a treatment of future trends and a brief summary