Establishment of stable and secretable Tatκ-GFP recombinant protein: a preliminary report of promoter methylation in 293t cell line

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

TAT Kappa (TATK) : a novel cell penetrating peptide for delivery of pluripotent proteins into target cells

Induced pluripotent stem cell (iPSC) holds a magnificent place in the medical revolution. Its emergence is expected to instigate development of novel therapies for regenerative medicine and treatment of malignant diseases. Moreover, iPSC usage also resolved a long-time ethical controversy on the usage of the embryo as a pluripotent stem cells source. Since Yamanaka’s iPSC discovery in 2006, several pieces of research have proven that the enforced expression of transcription factors Oct-3/4, KLF4, and Sox2 can induce the reprogramming of previously differentiated cells, to generate iPSC. However, the conventional method using viral vectors leads to genetic modification due to exogene integration and subsequently tumorigenicity, which is unsafe for clinical application. Therefore, our study utilised an improved novel protein transduction domain, trans-activator of transcription kappa (TAT), a synthetic TAT-HIV to deliver these transcription factors gene as an alternative method for iPSC generation via non-viral reprogramming. With this new strategy, we have established a stable clone of 293T cells expressing TATκ fusion proteins (TATκ-GFP, TATκ-KLF4, TATκ-Sox2, and TATκ-Oct-3/4) that expresses and secretes their respective cloned reprogramming proteins. These stable clones successfully transduced our target cell (U937) monocyte cell line. TATκ-GFP, a marker protein and fusion proteins TATκ-KLF4, TATκ-Sox2, and TATκ-Oct-3/4 transduced the targeted (U937) monocyte cell line, proving that this novel TATκ possesses an ability to translocate across the cell membrane. Morphological changes were successfully observed in U937 cells after 20 days of transduction, however the presence of bonifide iPSC colonies were unable to be elicited. This might be due to the incomplete reprogramming or insufficient duration of protein transduction to generate iPSC cells

Development of cell penetrating peptides for effective delivery of recombinant factors into target cells

The cell membrane is a protective layer that strictly controls the passage of molecules restricting the delivery of biomolecules such as drugs, oligonucleotides, peptides, and siRNA into the cells. This shortcoming has been overcome by the discovery of Cell-Penetrating Peptides (CPPs) that has undergone 30 years of evolution. To date, CPPs are largely modified to improve its efficacy and to suit the different delivery applications. The modes of CPPs penetration are still an unresolved mystery and requires further investigations to increase its effectiveness and to diversify its use. Despite having huge potential as a biomolecule carrier, CPPs also have some drawbacks. In this review, the natural and synthetic CPPs, the modifications that have been conducted on CPPs to improve its efficacy, its extended applications, modes of penetration and limitation as well as challenges will be discussed

Role of Olive Bioactive Compounds in Respiratory Diseases

Respiratory diseases recently became the leading cause of death worldwide, due to the emergence of COVID-19. The pathogenesis of respiratory diseases is centred around inflammation and oxidative stress. Plant-based alongside synthetic drugs were considered as therapeutics due to their proven nutraceutical value. One such example is the olive, which is a traditional symbol of the MedDiet. Olive bioactive compounds are enriched with antioxidant, anti-inflammatory, anticancer and antiviral properties. However, there are few studies relating to the beneficial effect of olive bioactive compounds on respiratory diseases. A vague understanding of its molecular action, dosage and bioavailability limits its usefulness for clinical trials about respiratory infections. Hence, our review aims to explore olive bioactive compound’s antioxidant, anti-inflammatory and antiviral properties in respiratory disease defence and treatment. Molecular insight into olive compounds’ potential for respiratory system protection against inflammation and ensuing infection is also presented. Olive bioactive compounds mainly protect the respiratory system by subsiding proinflammatory cytokines and oxidative stress

Effects of Hydroxytyrosol in Endothelial Functioning: A Comprehensive Review

Pharmacologists have been emphasizing and applying plant and herbal-based treatments in vascular diseases for decades now. Olives, for example, are a traditional symbol of the Mediterranean diet. Hydroxytyrosol is an olive-derived compound known for its antioxidant and cardioprotective effects. Acknowledging the merit of antioxidants in maintaining endothelial function warrants the application of hydroxytyrosol in endothelial dysfunction salvage and recovery. Endothelial dysfunction (ED) is an impairment of endothelial cells that adversely affects vascular homeostasis. Disturbance in endothelial functioning is a known precursor for atherosclerosis and, subsequently, coronary and peripheral artery disease. However, the effects of hydroxytyrosol on endothelial functioning were not extensively studied, limiting its value either as a nutraceutical supplement or in clinical trials. The action of hydroxytyrosol in endothelial functioning at a cellular and molecular level is gathered and summarized in this review. The favorable effects of hydroxytyrosol in the improvement of endothelial functioning from in vitro and in vivo studies were scrutinized. We conclude that hydroxytyrosol is capable to counteract oxidative stress, inflammation, vascular aging, and arterial stiffness; thus, it is beneficial to preserve endothelial function both in vitro and in vivo. Although not specifically for endothelial dysfunction, hydroxytyrosol safety and efficacy had been demonstrated via in vivo and clinical trials for cardiovascular-related studies