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.
Benzene exposure has been associated with hematotoxicity and leukemogenicity. However, the impact of benzene exposure on complex microenvironment of Hematopoetic Stem Cells (HSCs) niche, comprising of HSCs and lineage-specific progenitors remains elusive. Thus, a study on benzene-targeting HSCs niche could uncover mechanism linking benzene to HSCs niche alteration. This study evaluates the lineage-specific responses following exposure to a benzene metabolite, namely hydroquinone (HQ) in targeting HSCs and myeloid-committed progenitors. Freshly isolated murine bone marrow cells (BMCs) were exposed to HQ at series of concentrations (0 – 50 μM) for 24 hours; followed by cell viability analysis using MTT assay. Chromosomal aberration (CA) status was determined using karyotyping analysis. Expression of surface antigen for HSCs (Sca-1) was confirmed by flow cytometer. Lineage-specific myelotoxicity was studied using the colony-forming unit (CFU) assay for the following myeloid progenitors: CFU granulocyte /erythrocyte /macrophage /megakaryocyte (CFU-GEMM), CFU-granulocyte/macrophage (CFU-GM), CFU-granulocyte (CFU-G), CFU-macrophage (CFU-M), CFU-erythroid (CFU-E) and Burst-forming unit erythroid (BFU-E). HQ reduced (p
Pesticides and chemical fertilizers are widely used in agriculture to increase crop productivity among farmers. However, exposure to pesticides will give potential risk to human health. The aim of this study was to analyze the frequency of micronucleus (MN) and binucleus (BNu) formation in buccal cells from farmers who were exposed to pesticides using the MN assay. Buccal swabs were collected from the farmers in Tanjung Karang (n = 32) and Kelantan (n = 43) using wooden tongue depressor. A structured questionnaire was used to obtain demographic data of the farmers. Cytogenetic analysis was carried out by Acridin Orange (AO) staining 0.0025% (w/v). The frequency of MN and BNu as the biomarkers for cytogenetic damage was observed by using a fluorescence microscope. Comparison of frequency of MN and BNu is conducted in two areas namely Tanjung Karang, Selangor and Kelantan because of the agricultural activity and the type of pesticides used are different. Results showed that the frequencies of both MN and BNu among farmers in Tanjung Karang were significantly higher (p < 0.05) compared to farmers in Kelantan. Meanwhile, for the socio-demographic factors (age, smoking status, working period), MN and BNu frequencies among farmers in Tanjung Karang were also significantly higher (p < 0.05) as compared to farmers in Kelantan. While in the aspect of pesticide exposure, the frequencies of MN and BNu showed no significant difference between the frequency of pesticide spraying (p > 0.05) and the practices of PPE (Personal Protective Equipment) (p > 0.05). This may suggests that cytogenetic changes were not influenced by these factors. In addition, correlation study shows positive correlation between the frequency of MN with the pesticide exposure of farmers in Tanjung Karang (p > 0.05, r = 0.015) and Kelantan (p > 0.05, r = 0.0158). Besides, the frequency of BNu also has a positive correlation with the pesticide exposure among farmers in Tanjung Karang (p > 0.05, r = 0.036) and farmers in Kelantan (p > 0.05, r = 0.013). Hence, this present study demonstrated that exposure to pesticides increased the formation of MN and BNu among farmers and the prolonged use of pesticides may induce genotoxicity and DNA damage to human