In this study, tumorspheres were generated from TW06 nasopharyngeal carcinoma cell line and examined their expression of putative cancer stem-like cell surface markers and drug sensitivity. The rate of tumorsphere expansion from dissociated late passage TW06 tumorspheres (≥ passage 15) was higher than that from parental cells and dissociated 10-day-old (passage 0) tumorspheres. The expression of CD24 surface marker was lost in the generation of tumorspheres and the loss was reversible after differentiating the tumorspheres in monolayer culture conditions. Drug sensitivity assay showed that late passage tumorspheres were resistant to docetaxel and oxaliplatin treatment. Our data suggest that serially passaged tumorspheres possess the characteristics of CSCs that render them a suitable preclinical in vitro model for evaluating anticancer drug efficacy and elucidating the underlying mechanisms of drug resistance.
Neoplastic Stem Cells ; Nasopharyngeal carcinoma

Transforming growth factor-beta (TGFbeta) is present, predominantly in latent forms, in normal and malignant breast tissue. The mechanisms by which latent TGFbeta is activated physiologically remain largely an enigma. The objective of this study was to assess whether the proteases, cathepsin D and prostate specific antigen (PSA) could activate latent TGFbeta1 and TGFbeta2 in conditioned media of the hormone-dependent MCF-7 and hormone-independent MDA-MB-231 human breast cancer cell lines, newly purchased from ATCC. Both of the cell lines were seeded in 6-well plates 2 days prior to treatment with varying concentrations of cathepsin D and PSA. Active TGFbeta1 and TGFbeta2 in the media were then measured by ELISA after 4, 8, 24 and 72 hours of treatment. TGFbeta1 and TGFbeta2 mRNA expression of both cell lines were measured by RT-PCR to determine whether any increase in level of active TGFbeta1 and TGFbeta2 was due to increased production. There was a significant increase in only active TGFbeta2 levels in the MDA-MB-231 cell line with both treatments. Cathepsin D and PSA did not have any effect on TGFbeta1 and TGFbeta2 mRNA expression. Cathepsin D and PSA were unable to activate latent TGFbeta1 and TGFbeta2 in these two breast cancer cell lines. A constant level of TGFbeta2 mRNA in the control and treated MDA-MB-231 cells suggests that the increase in level of active TGFbeta2 was not a result of increased production but was likely to be due to activation by a mechanism independent of cathepsin D and PSA.
Transforming Growth Factor beta ; Cell Line ; Cathepsin D ; seconds ; public service announcement

Antibiotic susceptibility ; Staphylococcus aureus ; Community

Background: Immortalized human endothelial cells are widely used as in vitro models for debilitating conditions such as cancer, cardiovascular and ocular diseases. Human microvascular endothelial cell (HMEC-1) is immortalized via stable transfection with a gene encoding SV40 large antigen whilst telomerase-immortalized human microvascular endothelial (TIME) cells is immortalized by engineering the human telomerase catalytic protein (hTERT) into primary microvascular endothelial cells. Here, we established a three-dimensional (3D) spheroid invasion assay with HMEC-1 and TIME and compared the difference in their ability to invade through the collagen matrix in response to exogenous growth factors, namely vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Methods: TIME and HMEC-1 spheroids were embedded in a collagen matrix. The spheroids were stimulated with exogenous growth factors, namely VEGF (50ng/mL) and bFGF (200ng/mL). Twelve points of invasion length from a spheroid was measured using image analysis software, Image J. Three independent experiments were conducted and data was analysis by GraphPad Instat software, version 3.05. Results: TIME spheroid invasion was 16.5 fold higher with exogenous VEGF (50ng/mL) and bFGF (200ng/mL) treatment as compared to those cultured in complete growth medium only. In contrast, no significant difference was observed between HMEC-1 spheroids stimulated with and without exogenous growth factors, VEGF and bFGF. Conclusions: This is the first report on the establishment of a 3D-spheroid invasion assay with TIME cells. The requirement of VEGF and bFGF for TIME spheroids invasion is a novel finding. In addition, this assay offers an advantage over HMEC-1 for testing novel angiogenic agents since it is not affected by endogenously secreted growth factors.

Signal transduction pathways are constitutively expressed in leukaemic cells resulting in aberrant survival of the cells. It is postulated that in cells of chemo-sensitive patients, chemotherapy induces apoptotic signals leading to cell death while survival signals are maintained in cells of chemo-resistant patients. There is very little information currently, on the expression of these mediators in patients immediately after chemotherapy initiation. We examined the expression pattern of proinflammatory cytokines, signaling molecules of the PI3K and MAPK pathways molecules and death receptor, DR5 on paired samples at diagnosis and during chemotherapy in acute myeloid leukaemia patients treated with cytosine arabinoside and daunorubicin. The results were correlated with remission status one month after chemotherapy. We found that in chemo-sensitive patients, chemotherapy significantly increased the percentage of cases expressing TNF-alpha (p = 0.025, n = 9) and IL-6 (p = 0.002, n = 11) compared to chemo-resistant cases. We also observed an increased percentage of chemo-sensitive cases expressing DR5 and phosphorylated p38, and Jnk. Thus, expression of TNF-alpha, IL-6, DR5, phospho-p38 and phospho-Jnk may regulate cell death in chemo-sensitive cases. In contrast, a significantly higher percentage of chemo-resistant cases expressed phospho-Bad (p = 0.027, n = 9). IL-beta and IL-18 were also found to be higher in chemo-resistant cases at diagnosis and during chemotherapy. Thus, expression of various cellular molecules in leukaemic blasts during chemotherapy may be useful in predicting treatment outcome. These cellular molecules may also be potential targets for alternative therapy.

Introduction: Current prognostic markers have improved survival prediction, however, it has not advanced treatment strategies. Gene expression profiling may identify biological markers suitable as therapeutic targets. Leukaemia stem cell is associated with adverse outcome, however, its biological characteristics are still being investigated. We observed higher in vitro cell viability in acute myeloid leukaemia (AML) samples with poor prognosis, which may be stem cell related. Objective: The objective of this study was to profile highly expressed genes in an AML sample of poor prognosis/high viability and compare with a sample of good prognosis/low viability. Method: Subtractive hybridization was performed on two AML samples with high blast counts (>80%), a poor prognosis, PP (disease free survival, DFS<12 months) and a good prognosis, GP (DFS>12 months) sample. The PP sample had higher CD34+ counts (73% vs 46%) and higher cell viability than the GP sample. cDNA libraries were subsequently cloned and sequenced. Results: cDNA subtracted from the PP samples was identified as genes active during fetal/embryonic development (LCOR, CNOT1, ORMDL1), HOX- related genes (HOXA3, PBX3, SF3B1), hematopoiesis (SELL, IL-3RA) and aerobic glycolysis/hypoxia (PGK1, HIGD1A) -associated genes. Majority of GP clones isolated contained genes involved in oxidative phosphorylation, OXPHOS (COXs, ATPs, MTND4 and MTRNR2), protein synthesis (including ribosomal proteins, initiating and elongation factors), chromatin remodeling (H2AFZ, PTMA), cell motility (MALAT1, CALM2, TMSB4X), and mitochondria (HSPA9, MPO) genes. Conclusion: Thus, the PP sample exhibited stem cell-like features while the GP sample showed cells at a high level of cell activity. These genes are potential prognostic markers and targets for therapy.
Leukemia, Myeloid, Acute

Introduction: The phenotype and genotype of cancer cells portray hallmarks of cancer which may have clinical value. Cancer cell lines are ideal models to study and confirm these characteristics. We previously established two subtracted cDNA libraries with differentially expressed genes from an acute myeloid leukaemia patient with poor prognosis (PP) and good prognosis (GP). Objective: To compare gene expression of the leukaemia associated genes with selected biological characteristics in leukaemia cell lines and normal controls. Methodology: Expression of 28 PP genes associated with early fetal/embryonic development, HOX-related genes, hematopoiesis and aerobic glycolysis/ hypoxia genes and 36 GP genes involved in oxidative phosphorylation, protein synthesis, chromatin remodelling and cell motility were examined in B-lymphoid (BV173, Reh and RS4;11) and myeloid (HL-60, K562) leukaemia cell lines after 72h in culture as well as peripheral blood mononuclear cells from healthy controls (N=5) using semi-quantitative polymerase chain reaction (PCR) method. Cell cycle profiles were analysed on flow cytometry while MTT cytotoxicity assay was used to determine drug resistance to epirubicin. Results: Genes expressed significantly higher in B-lymphoid leukaemia cell lines compared to healthy controls were mostly of the GP library i.e. oxidative phosphorylation (3/10), protein synthesis (4/11), chromatin remodelling (3/3) and actin cytoskeleton genes (1/5). Only two genes with significant difference were from the PP library. Cancer associated genes, HSPA9 and PSPH (GP library) and BCAP31 (PP library) were significantly higher in the B-lymphoid leukemia cell lines. No significant difference was observed between myeloid cell lines and healthy controls. This may also be due heterogeneity of cell lines studied. PBMC from healthy controls were not in cell cycle. G2/M profiles and growth curves showed B-lymphoid cells just reaching plateau after 72 hour culture while myeloid cells were declining. IC50 values from cytotoxicity assay revealed myeloid cell lines had an average 13-fold higher drug resistance to epirubicin compared to B-lymphoid cell lines. Only CCL1, was expressed at least two-fold higher in myeloid compared to B-lymphoid cell lines. In contrast, MTRNR2, EEF1A1, PTMA, HLA-DR, C6orf115, PBX3, ENPP4, SELL, and IL3Ra were expressed more than 2-fold higher in B-lymphoid compared to myeloid cell lines studied here. Conclusion: Thus, B-lymphoid leukaemia cell lines here exhibited active, proliferating characteristics closer to GP genes. Higher expression of several genes in B-lymphoid compared to myeloid leukaemia cell lines may be useful markers to study biological differences including drug resistance between lineages.
Neoplasms

The Life Support Course for Nurses (LSCN) equips nurses with the resuscitation skills to be first responders in in-hospital cardiac arrests. Seventeen years after the initiation of the LSCN, a confidential cross-sectional Qualtrics™ survey was conducted in May 2016 on LSCN graduands to assess the following: confidence in nurse-initiated resuscitation post-LSCN; defibrillation experience and outcomes; and perceived barriers and usefulness of the LSCN. The majority of respondents reported that the course was useful and enhanced their confidence in resuscitation. Skills retention can be enhanced by organising frequent team-based resuscitation training. Resuscitation successes should be publicised to help overcome perceived barriers.

Humans ; Urology ; Pandemics ; COVID-19/epidemiology* ; SARS-CoV-2

INTRODUCTIONWith the advent of antibiotics, complications of otitis media have become less common. It is crucial for physicians to recognise otitis media and treat its complications early. Herein, we present our institution's experience with patients who required emergency surgical intervention for complications of otitis media.

METHODSData on patients who underwent emergency surgery for complications of otitis media from 2004 to 2011 was retrieved from the archives of the Department of Otolaryngology, Changi General Hospital, Singapore.

RESULTSA total of 12 patients (10 male, two female) underwent emergency surgery for complications of otitis media. The median age of the patients was 25 years. Otalgia, otorrhoea, headache and fever were the main presenting symptoms. Extracranial complications were observed in 11 patients, and six patients had associated intracranial complications. The primary otologic disease was acute otitis media in six patients, chronic otitis media without cholesteatoma in three patients and chronic otitis media with cholesteatoma in three patients. Mastoidectomy and drainage of abscess through the mastoid, with insertion of grommet tube, was the main surgical approach. Two patients required craniotomy. The mean length of hospital stay was 16.2 days and the mean follow-up period was 16.3 months. Five patients had residual conductive hearing loss; two patients with facial palsy had full recovery.

CONCLUSIONOtitis media can still result in serious complications in the post-antibiotic era. Patients with otitis media should be monitored, and prompt surgical intervention should be performed when necessary to attain good outcomes.

Adult ; Audiometry ; Chronic Disease ; Facial Paralysis ; complications ; Female ; Fever ; complications ; Hospitals ; Humans ; Length of Stay ; Male ; Middle Aged ; Otitis Media ; complications ; diagnostic imaging ; surgery ; Retrospective Studies ; Singapore ; Treatment Outcome ; Young Adult