Introduction: Human leukocyte antigens (HLA) are a group of unique transmembrane glycoproteins that are ex-pressed on the surface of virtually all types of cells within the human body. These molecules are encoded by a set of highly polymorphic gene sequences known also as the major histocompatibility complex (MHC) and play an essential role in the presentation of antigenic peptides to immune cells for recognition and response. In recent years, various HLA alleles have been found to be associated with different autoimmune and inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE) and allergic rhinitis. Identification of these alleles via HLA typing is necessary for initial screening and diagnosis purposes. Besides that, HLA typing is also used to determine compatibility matching between a donor and a recipient for tissue/organ transplantations in order to prevent graft rejection. Therefore, good quality and quantity of genomic DNA is required. In most scenarios, peripheral blood is chosen as the most reliable source of DNA for analysis, however this approach is seen as invasive and may cause pain and anxiety among the patients, particularly young children and weak subjects. Hence, derivation of genomic DNA from buccal cells as an alternative source material is becoming increasingly popular, especially in PCR-based genetic assays. Some of the most commonly described methods to collect buccal cells include using oral swabs, cytological brushes, mouthwashes and treated cards. Each technique yields varying quantities of DNA with diverse purity levels. In this study, we aim to evaluate the amount and purity of genomic DNA extracted from buccal swabs and brushes as well as blood for screening of selected HLA class II alleles. Methods: Cheek cell samples were col-lected using sterile foam tipped buccal swabs (Whatman) and buccal collection brushes (Gentra Puregene) whereas peripheral blood samples were withdrawn following routine venipuncture techniques. All samples were subjected to DNA extraction according to modified commercial kit protocols. Screening of selected HLA-DRB1 alleles was con-ducted via PCR with sequence-specific primers as established by Bunce et al. 1995. Results: There was no significant difference (p > 0.05) in the total DNA yield obtained from blood and buccal swab samples, which were 17.57μg (± 8.66) and 13.28μg (± 4.81), respectively. All samples exhibited similar 260/280 ratios of about ~1.80 (p > 0.05). However, buccal brush samples contributed the least amount of DNA (0.29μg, ± 0.12) compared to other sources (p < 0.05). The pure genomic DNA isolated from both blood and buccal swab samples were successfully typed for low resolution HLA-DRB1 alleles. Conclusion: Buccal swabs provide good quantity and quality of DNA for screening of HLA alleles with high accuracy and thus can be utilized as a non-invasive substitute for venipuncture.
Induced pluripotent stem cells (iPSC) is a novel technology useful for therapeutic and research applications. To date, iPSCs
is produced through genetic modification that can promote mutation; making it harmful for therapeutic use. Therefore,
application of non-genetic modification through direct delivery of recombinant proteins aided by protein transduction
domain (PTD) enable a safer production of iPSC. This study is aimed to establish a stable production of secretable
recombinant protein via recombination of green fluorescence protein (GFP) and a novel PTD peptide, namely TATκ-GFP.
293Tcell line was transfected with 20 µg/ml of TATκ-GFP plasmid and the stably transfected 293T cells were then cultured
for 54 days to determine the stability of expression and secretion of TATκ-GFP recombinant protein in prolonged culture.
Methylation at the CMV promoter of the TATκ-GFP plasmid was investigated following treatment of transfected cells with
3 µM/mL of demethylation agent, namely 5-Azacytidine for 72 h in three cycles. Flow cytometry analysis demonstrated
a transfection efficiency of 9.33% and successful secretion of TATκ-GFP proteins into the culture medium as analysed by
Western blot at 72 h post-transfection. However, the transfected cells exhibited a decreasing level of GFP expression and
secretion following prolonged culture with notable stability that only sustained for two weeks. 5-Azacytidine-treated cells
showed a slight increase of GFP expression compared to non-treated control, suggesting possible promoter methylation
which could cause instability of TATκ-GFP expression. Conclusively, promoter methylation should be considered for future
establishment of iPSCs as it could inhibit stable expression and secretion of recombinant proteins.
The aim of this study to determine the numbers of CD34+ cells and total nucleated cell (TNC) in umbilical cord blood (UCB)
collected from pregnant mothers with gestational diabetes mellitus (GDM) and preeclampsia (PE), following statistical
analysis of both maternal and perinatal factors which affect UCB parameters. Most of studies explored the influence of
obstetric factors on the number of UCB cell collection and only a few looked at the effects on UCB haematopoietic stem
cell (UCB-HSC) of common disorders complicating pregnancy. A total of 112 UCB samples (32 PE, 42 GDM and 38 nondiseased) were collected. CD34+ cell and NC count were enumerated using FACS Calibur. The TNC and CD34+ cells were
significantly reduced in both PE and GDM groups as compared to the control group. The PE group shows significantly
lower birth weight and higher BP which led to a lower UCB volume and CD34+ count. Gestational age shows significant
correlation with nucleated cell count (NCC) and TNC. GDM group shows significantly lower systolic BP, NCC and TNC count,
including low placental weight and birth weight. Conclusively, some obstetrics factors have significant influences to the
numbers and quality of UCB-HSC in both PE and GDM groups, which could guide in the selection criteria for CB banking.
DNA-templated silver nanocluster (AgNC), a new promising fluorescence probe has gained importance in biosensing and bioimaging in recent years. We employed a label-free AgNC to detect an intracellular transcription factor known as forkhead box p3 (FOXP3), which is the master regulator of regulatory T cells (Tregs) suppressive function. We developed an optimized method for the detection of messenger ribonucleic acid (mRNA) of FOXP3 by hybridizing AgNC and G-rich to the target FOXP3 mRNA of a MCF-7 cells. MCF-7 cells are chosen as a model as it readily expresses FOXP3. The hybridized samples were examined with UV illuminator and further verified with fluorescence spectroscopy, fluorescence microscope and flow cytometry. The successful hybridization of a three-way junction with AgNC, G-rich and mRNA FOXP3 target generated an improved fluorescence intensity with a spectral shift. We have successfully delivered the green fluorescing AgNC and G-rich into MCF-7 cells, producing a shift to red fluorescing cells corroborated by flow cytometry results. In summary, our approach enables the detection of intracellular FOXP3 nucleic acid and holds considerable potential in establishing a non-lethal intracellular detection system which would be crucial for the isolation of regulatory T-cells (Tregs) when combined with other cell surface markers.