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Fulltext Development and evaluation of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus in human, monkey and mosquito samples

Ummul Haninah A, Vasan SS, Ravindran T, Chandru A, Lee HL, Shamala Devi S

Trop Biomed, 2010 Dec;27(3):611-23.
PMID: 21399603 MyJurnal

This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/μl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.

Matched MeSH terms: Staining and Labeling/methods
Metabolic glycan labeling and chemoselective functionalization of native biomaterials

Ren X, Evangelista-Leite D, Wu T, Rajab TK, Moser PT, Kitano K, et al.

Biomaterials, 2018 11;182:127-134.
PMID: 30118980 DOI: 10.1016/j.biomaterials.2018.08.012

Decellularized native extracellular matrix (ECM) biomaterials are widely used in tissue engineering and have reached clinical application as biomesh implants. To enhance their regenerative properties and postimplantation performance, ECM biomaterials could be functionalized via immobilization of bioactive molecules. To facilitate ECM functionalization, we developed a metabolic glycan labeling approach using physiologic pathways to covalently incorporate click-reactive azide ligands into the native ECM of a wide variety of rodent tissues and organs in vivo, and into the ECM of isolated rodent and porcine lungs cultured ex vivo. The incorporated azides within the ECM were preserved after decellularization and served as chemoselective ligands for subsequent bioconjugation via click chemistry. As proof of principle, we generated alkyne-modified heparin, immobilized it onto azide-incorporated acellular lungs, and demonstrated its bioactivity by Antithrombin III immobilization and Factor Xa inhibition. The herein reported metabolic glycan labeling approach represents a novel platform technology for manufacturing click-reactive native ECM biomaterials, thereby enabling efficient and chemoselective functionalization of these materials to facilitate tissue regeneration and repair.

Matched MeSH terms: Staining and Labeling/methods
Fulltext Computer-assisted quantification of CD3+ T cells in follicular lymphoma

Abas FS, Shana'ah A, Christian B, Hasserjian R, Louissaint A, Pennell M, et al.

Cytometry A, 2017 06;91(6):609-621.
PMID: 28110507 DOI: 10.1002/cyto.a.23049

The advance of high resolution digital scans of pathology slides allowed development of computer based image analysis algorithms that may help pathologists in IHC stains quantification. While very promising, these methods require further refinement before they are implemented in routine clinical setting. Particularly critical is to evaluate algorithm performance in a setting similar to current clinical practice. In this article, we present a pilot study that evaluates the use of a computerized cell quantification method in the clinical estimation of CD3 positive (CD3+) T cells in follicular lymphoma (FL). Our goal is to demonstrate the degree to which computerized quantification is comparable to the practice of estimation by a panel of expert pathologists. The computerized quantification method uses entropy based histogram thresholding to separate brown (CD3+) and blue (CD3-) regions after a color space transformation. A panel of four board-certified hematopathologists evaluated a database of 20 FL images using two different reading methods: visual estimation and manual marking of each CD3+ cell in the images. These image data and the readings provided a reference standard and the range of variability among readers. Sensitivity and specificity measures of the computer's segmentation of CD3+ and CD- T cell are recorded. For all four pathologists, mean sensitivity and specificity measures are 90.97 and 88.38%, respectively. The computerized quantification method agrees more with the manual cell marking as compared to the visual estimations. Statistical comparison between the computerized quantification method and the pathologist readings demonstrated good agreement with correlation coefficient values of 0.81 and 0.96 in terms of Lin's concordance correlation and Spearman's correlation coefficient, respectively. These values are higher than most of those calculated among the pathologists. In the future, the computerized quantification method may be used to investigate the relationship between the overall architectural pattern (i.e., interfollicular vs. follicular) and outcome measures (e.g., overall survival, and time to treatment). © 2017 International Society for Advancement of Cytometry.

Matched MeSH terms: Staining and Labeling/methods
Detection and differentiation of velogenic and lentogenic Newcastle disease viruses using SYBR Green I real-time PCR with nucleocapsid gene-specific primers

Tan SW, Ideris A, Omar AR, Yusoff K, Hair-Bejo M

J Virol Methods, 2009 Sep;160(1-2):149-56.
PMID: 19447142 DOI: 10.1016/j.jviromet.2009.05.006

SYBR Green I real-time PCR was developed for detection and differentiation of Newcastle disease virus (NDV). Primers based on the nucleocapsid (NP) gene were designed to detect specific sequence of velogenic strains and lentogenic/vaccine strains, respectively. The assay was developed and tested with NDV strains which were characterized previously. The velogenic strains were detected only by using velogenic-specific primers with a threshold cycle (C(t)) 18.19+/-3.63 and a melting temperature (T(m)) 86.0+/-0.28 degrees C. All the lentogenic/vaccine strains, in contrast, were detected only when lentogenic-specific primers were used, with the C(t) value 14.70+/-2.32 and T(m) 87.4+/-0.21 degrees C. The assay had a dynamic detection range which spans over a 5log(10) concentration range, 10(9)-10(5) copies of DNA plasmid/reaction. The velogenic and lentogenic amplifications showed high PCR efficiency of 100% and 104%, respectively. The velogenic and lentogenic amplifications were highly reproducible with assay variability 0.45+/-0.31% and 1.30+/-0.65%, respectively. The SYBR Green I real-time PCR assay detected successfully the virus from tissue samples and oral swabs collected from the velogenic and lentogenic NDV experimental infection, respectively. In addition, the assay detected and differentiated accurately NDV pathotypes from suspected field samples where the results were in good agreement with both virus isolation and analysis of the fusion (F) cleavage site sequence. The assay offers an attractive alternative method for the diagnosis of NDV.

Matched MeSH terms: Staining and Labeling/methods
Detection of influenza A and B neutralizing antibodies in vaccinated ferrets and macaques using specific biotin-streptavidin conjugated antibodies

Sirskyj D, Weltzin R, Golshani A, Anderson D, Bozic J, Diaz-Mitoma F, et al.

J Virol Methods, 2010 Feb;163(2):459-64.
PMID: 19913054 DOI: 10.1016/j.jviromet.2009.11.014

Several critical factors of an influenza microneutralization assay, utilizing a rapid biotin-streptavidin conjugated system for detecting influenza virus subtypes A and B, are addressed within this manuscript. Factors such as incubation times, amount of virus, cell seeding, sonication, and TPCK trypsin were evaluated for their ability to affect influenza virus neutralization in a microplate-based neutralization assay using Madin-Darby canine kidney (MDCK) cells. It is apparent that the amount of virus used in the assay is the most critical factor to be optimized in an influenza microneutralization assay. Results indicate that 100xTCID(50) of influenza A/Solomon Islands/03/2006 (H1N1) virus overloads the assay and results in no, to low, neutralization, in both ferret and macaque sera, respectively, whereas using 6xTCID(50) resulted in significantly improved neutralization. Conversely, strong neutralization was observed against 100xTCID(50) of B/Malaysia/2506/04 virus. In this manuscript the critical factors described above were optimized and the results indicate that the described biotin-streptavidin conjugated influenza microneutralization assay is a rapid and robust method for detecting the presence of functional, influenza virus-neutralizing antibodies.

Matched MeSH terms: Staining and Labeling/methods
Fulltext Antiproliferative property and apoptotic effect of xanthorrhizol on MDA-MB-231 breast cancer cells

Cheah YH, Nordin FJ, Tee TT, Azimahtol HL, Abdullah NR, Ismail Z

Anticancer Res, 2008 Nov-Dec;28(6A):3677-89.
PMID: 19189649

Xanthorrhizol is a natural sesquiterpenoid compound isolated from the rhizome of Curcuma xanthorrhizza Roxb (Zingerberaceae). Recent studies of xanthorrhizol in cell cultures strongly support the role of xanthorrhizol as an antiproliferative agent. In our study, we tested the antiproliferative effect of xanthorrhizol using different breast cancer cell lines. The invasive breast cancer cell line, MDA-MB-231, was then selected for further investigations. Treatment with xanthorrhizol caused 50% growth inhibition on MDA-MB-231 cells at 8.67 +/- 0.79 microg/ml as determined by sulforhodamine B (SRB) assay. Hoechst 33258 nuclear staining assay showed the rate of apoptosis of MDA-MB-231 cells to increase in response to xanthorrhizol treatment. Immunofluorescence staining using antibody MitoCapture and fluorescein isothiocyanate (FITC)-labeled cytochrome c revealed the possibility of altered mitochondrial transmembrane potential and the release of cytochrome c respectively. This was further confirmed by Western-blotting, where cytochrome c was showed to migrate from mitochondrial fraction to the cytosol fraction of treated MDA-MB-231 cells. Caspase activity assay showed the involvement of caspase-3 and caspase-9, but not caspase-6 or caspase-8 in MDA-MB-231 apoptotic cell death. Subsequently, cleavage of PARP-1 protein is suggested. These data suggest treatment with xanthorrhizol modulates MDA-MB-231 cell apoptosis through the mitochondria-mediated pathway subsequent to the disruption of mitochondrial transmembrane potential, release of cytochrome c, activation of caspase-3 and caspase-9, and the modulation of PARP-1 protein.

Matched MeSH terms: Staining and Labeling/methods
Temporal proteomic profiling of Chlamydia trachomatis-infected HeLa-229 human cervical epithelial cells

Tan GM, Lim HJ, Yeow TC, Movahed E, Looi CY, Gupta R, et al.

Proteomics, 2016 05;16(9):1347-60.
PMID: 27134121 DOI: 10.1002/pmic.201500219

Chlamydia trachomatis is the leading causative agent of bacterial sexually transmitted infections worldwide which can lead to female pelvic inflammatory disease and infertility. A greater understanding of host response during chlamydial infection is essential to design intervention technique to reduce the increasing incidence rate of genital chlamydial infection. In this study, we investigated proteome changes in epithelial cells during C. trachomatis infection by using an isobaric tags for relative and absolute quantitation (iTRAQ) labeling technique coupled with a liquid chromatography-tandem mass spectrometry (LC-MS(3) ) analysis. C. trachomatis (serovar D, MOI 1)-infected HeLa-229 human cervical carcinoma epithelial cells (at 2, 4 and 8 h) showed profound modifications of proteome profile which involved 606 host proteins. MGST1, SUGP2 and ATXN10 were among the top in the list of the differentially upregulated protein. Through pathway analysis, we suggested the involvement of eukaryotic initiation factor 2 (eIF2) and mammalian target of rapamycin (mTOR) in host cells upon C. trachomatis infection. Network analysis underscored the participation of DNA repair mechanism during C. trachomatis infection. In summary, intense modifications of proteome profile in C. trachomatis-infected HeLa-229 cells indicate complex host-pathogen interactions at early phase of chlamydial infection.

Matched MeSH terms: Staining and Labeling/methods
Comparison of the sensitivity and specificity of p16/Ki-67 dual staining and HPV DNA testing of abnormal cervical cytology in the detection of histology proven cervical intraepithelial neoplasia grade 2 and above (CIN 2+)

Tay TKY, Lim KL, Hilmy MH, Thike AA, Goh ST, Song LH, et al.

Malays J Pathol, 2017 Dec;39(3):257-265.
PMID: 29279588

INTRODUCTION: Human papillomavirus (HPV) testing is used as a means of triaging cervico-vaginal smears with low grade squamous abnormalities or as part of co-testing with cytology. While HPV testing has a high sensitivity, it has a low specificity in detecting cervical intraepithelial neoplasia grade 2 and above (CIN 2+) leading to unnecessary colposcopy referrals. We investigate the accuracy of the p16/Ki-67 dual immunocytochemical stain in determining the presence of CIN 2+ lesions on histology and its potential as a superior biomarker for triage.
METHODS: Liquid based cervico-vaginal cytology specimens with squamous abnormalities and corresponding histology from 97 women with subsequent colposcopy and biopsy were included. The specimens were then subjected to the dual stain and Roche Cobas 4800 multiplex real time PCR HPV DNA testing. The sensitivity and specificity of the dual stain and HPV testing were calculated using CIN 2+ on histology as a reference standard.
RESULTS: The sensitivity and specificity of the dual stain in detecting histology proven CIN 2+ was 93.7% and 76.5% while HPV testing was 85.7% and 14.7% respectively. Of the 44 women with ASCUS or LSIL on cytology, the dual stain also reduced the number of unnecessary colposcopy referrals from 27 to 7 when used as a triage marker compared to HPV testing.
CONCLUSION: p16/Ki-67 dual stain was more sensitive and specific than HPV testing in determining the presence of CIN 2+ on histology. It could triage low grade cervico-vaginal specimens more effectively and potentially help women avoid unnecessary colposcopies. Future studies are needed to further evaluate its role in cervical cancer screening programmes.

Matched MeSH terms: Staining and Labeling/methods
Expression of multidrug resistance (MDR) proteins and in vitro drug resistance in acute leukemias

Fazlina N, Maha A, Jamal R, Zarina AL, Cheong SK, Hamidah H, et al.

Hematology, 2007 Feb;12(1):33-7.
PMID: 17364990

The expression of the multidrug resistance (MDR) proteins may influence the outcome of treatment in patients with acute leukemia. The aim of this study was to determine the IC50 of cytotoxic drugs (cytosine arabinoside, ara-C and daunorubicin, dnr) using the in vitro 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt (MTS) assay method. A total of 82 newly diagnosed acute leukemia cases (43 adult myeloid leukaemia, AML cases and 39 acute lymphoblastic leukaemia, ALL cases) and 16 relapsed cases (8 AML cases and 8 ALL cases) were studied. The MTS assay was performed using two cytotoxic drugs, dnr and ara-C. Cells were incubated with different concentrations of drugs for 4 days and the IC50 was extrapolated from the viability curve. In newly diagnosed cases, we found that childhood ALL samples showed higher IC50 values of dnr (0.040 +/- 2.320) compared to adult AML samples (0.021 +/- 0.158). In contrast, newly diagnosed adult AML samples showed higher IC50 values of ara-C (0.157 +/- 0.529) compared to childhood ALL samples (0.100 +/- 2.350). In relapsed cases, two samples of childhood ALL showed IC50 values of dnr (0.910 +/- 1.760) and ara-C (1.310 +/- 2.390), which was higher compared to childhood AML samples (0.129 +/- 0.214 and 0.210 +/- 0.003, respectively). However, there was no correlation between IC50 values of these drugs tested with clinical outcome. In conclusion, we found that MTS assay is an easy, rapid and non laborious method to study in vitro drug resistance in acute leukaemia cases.

Matched MeSH terms: Staining and Labeling/methods