Browsing by Author "Mohd Azri Abd Jalil"

When the skin is injured, it would compromise the immune system as well as body’s homeostasis. Wound that fail to progress into the normal stage of healing is being recognized as delayed acute wound and chronic wound that could worsen the body condition. Honey is a well establish treatment for wound healing due to its antimicrobial, antioxidants, anti-inflammatory and high sugar contents. First of all, the stingless bee honey was evaluated via in vitro in term of antimicrobial and anti-inflammatory properties. 40% w/v concentration of honey displayed a good antimicrobial property against Staphylococcus aureus colonies since the honey able to inhibit their growth. In addition, the honey did not reduce the proinflammatory cytokines produced by LPS-activated macrophages. It is due to the immunomodulatory properties of honey that react towards the cytokines depending on the microenvironment of the wound. From the in vitro evaluation, 40% w/v concentration of honey was selected to be incorporated with hydrogel. Then, stingless bee-based hydrogel formulation was developed and characterized. Response surface model (RSM) which is consist of factorial design and central composite design was used to develop and optimized the hydrogel. Then, the characterization of the formulation was studied in term of hydrophilicity, polymerization, water vapor transmission rate (WVTR), rheological, drug release, microbial limit test, antimicrobial, stability and interaction between honey and the excipient by using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR). Formulation 3 displayed a good reaction towards the selected response. Formulation 3 hydrogel has a good swelling ability which could provide a good moisturizing condition to the wound. Formulation 3 exhibited a good WVTR where the value is lower than control formulation. A higher WVTR cause the wound to dry rapidly. In rheological properties, Formulation 3 displayed a clear LVR line while in ATR-FTIR test, there was no substantial interaction observed in comparison with blank hydrogel. In addition, the release profile in Formulation 3 followed diffusion-controlled mechanism where the delivery of the honey is consistent throughout the time. Then, in term of microbial activity, Formulation 3 exhibited a good antimicrobial property and free from pathogenic microorganism. Finally, from the accelerated stability data, Formulation 3 was stable throughout storage period. For in vivo study, the blank and honey hydrogel were safe to use on the skin of the rabbit. The healing in honey hydrogel treated group was significantly (p<0.05) faster than no treatment group as shown in wound closure percentage and histological assessment. In honey hydrogel treated group, the amount of collagen deposited is abundant and the structure of fibroblast is well organized, this indicated at the end of the animal study, the wound has entered the remodelling phase compared with the no treatment group that still in proliferation phase. Based on all of these results, there are a lot of promising properties of stingless bee honey-based hydrogel such as good hydrophilicity, elasticity, microbial safety, reliable release profile as well as efficacy in wound healing treatment. Hence, it can be concluded that stingless bee honey-based hydrogel has a high potential to be a good wound dressing.

Hepatocyte Nuclear Factor 1 Beta (HNF1B) is a DNA-binding transcription factor that is essential for normal kidney development and is expressed in all tubular epithelial cells composing the nephrons and collecting ducts where it controls the expression of genes involved in membrane transport, cell differentiation, and metabolism. In this study, CRISPR/Cas9 technology was utilised to generate HNF1B gene knockout in HEK 293T cells, to investigate the effects of HNF1B gene loss. To achieve this, HEK 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin (10,000 IU). Two guide RNAs (sgRNAs) targeting HNF1B gene were designed using the CRISPick web tool, and DNA breaks were repaired through non-homologous end-joining (NHEJ) pathway. The sgHNF1B was cloned into the pKLV-U6gRNA(BbsI)-PGKhygro2ABFP vector, transformed into E. coli and successful cloning was then validated using Sanger sequencing. Post-transduction of Cas9 lentiviral construct, cells were selected with 30 μg/mL Blasticidin antibiotic, and the edited cells were isolated. Subsequently, these cells (containing Cas9) were transduced with sgHNF1B lentivirus and further selected using 700 μg/mL Hygromycin selection for 6 days and continue with western blot to validate the protein expression. The western blot band intensity analysis indicates that HNF1B protein expression is reduced in both knockout cell lines, KO1 (60%) and KO2 (34%), compared to untransduced control (100%). The observed reduction in protein expression levels suggests that the knockout is heterozygous and not full knock out. This is likely due to a mixed cell population, where some cells are fully edited (bi-allelic KO), others are partially edited (monoallelic) and some may remain unedited resulting in intermediate levels of protein expression when analysed collectively. The HNF1B gene knockout cells were successfully compared with untransduced control cells and further validated using western blot analysis confirming the HNF1B gene knockout at the protein level. In conclusion, this study had successfully generated heterozygous HNF1B CRISPR-knockout models of kidney dysfunction as a proof-of-concept that gene function can be altered to treat kidney diseases. In addition, this study also highlights the utility of genome editing as a powerful tool for elucidating gene function and effectively modelling human pathophysiological conditions. Through this CRISPR/Cas9 technology, valuable insights are offered to drive advancements in personalized medicine and sustainable healthcare solution. Such innovations address key biomedical challenges and advance scientific progress to improve the well-being of both individuals and the planetary health. Keywords: CRISPR/Cas9, Gene editing, HEK 293T, HNF1B, sgRNA cloning, kidney