METHODS AND ANALYSIS: A three-step approach consensus approach will be adopted for this study for the development of a validated medical curriculum guideline on AMR. A preliminary curriculum for the programme will be drafted from reviews of published literature including syllabi as well as national and international guidelines. A total of 26 potential sources were found to be relevant, and selected for this study. Subsequently, the drafted curriculum will be subjected for validation via online surveys by various infectious disease experts. Finally, a Delphi technique will be employed to obtain consensus on heterogeneous findings to the revised curriculum. The quantitative and qualitative responses will be analysed and discussed among the panel of researchers.
ETHICS AND DISSEMINATION: This study protocol has been approved by the Institute of Health Sciences Research Ethics Committee of Universiti Brunei Darussalam (Reference: UBD/PAPRSBIHSREC/2020/124). Informed consent declaration will be collected prior to data collections as indication of agreement of participation in the study. Results will be made available to medical educators and also researchers on AMR and stewardship. The results also will be disseminated at feedback sessions to officers at Ministry of Health and Ministry of Education, Brunei Darussalam.
METHODS: The recombinant BipD protein was produced, followed by isolation of BipD-specific aptamers using Systematic Evolution of Ligands by EXponential enrichment. The binding affinity and specificity of the selected aptamers were evaluated using Enzyme-Linked Oligonucleotide Assay.
RESULTS: The fifth SELEX cycle showed a notable enrichment of recombinant BipD protein-specific aptamers. Sequencing analysis identified two clusters with a total of seventeen distinct aptamers. AptBipD1, AptBipD13, and AptBipD50 were chosen based on their frequency. Among them, AptBipD1 exhibited the highest binding affinity with a Kd value of 1.0 μM for the recombinant BipD protein. Furthermore, AptBipD1 showed significant specificity for B. pseudomallei compared to other tested bacteria.
CONCLUSION: AptBipD1 is a promising candidate for further development of reliable, affordable, and efficient point-of-care diagnostic tests for melioidosis.
Methods: The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3'end of the reporter gene and the VP2 start sequence to allow co-translational 'cleavage' of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones.
Results: Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing.
Conclusion: NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication.
METHODS: The antioxidant properties were analysed using 2,2'-diphenyl-1 picrylhydrazyl (DPPH), ferric reducing antioxidant potential (FRAP) and 2,2'-azinodi 3-ethylbenthiazolinesulfonate (ABTS) assays. Cytotoxicity was assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test and the induction of apoptosis was observed via caspase-3/7 activity assay. The identification of phenolic compounds was done via ultra-high-performance-liquid chromatography coupled to mass spectrometer (UHPLC-MS/MS).
RESULTS: The antioxidant analysis exhibited: the half inhibitory concentration (IC50) of DPPH inhibition, 54.10 (SD = 4.51) μg/mL; the FRAP value, 30.07 (SD = 0.93) mM TEQ/100 g; and the ABTS value, 131.79 (SD = 8.73) mg TEQ/100 g. Cinnamic acid was the most abundant phenolic compound, followed by maleic acid and salicylic acid. The IC50 for ORL115 and ORL188 were 35.51 mg/mL and 43.54 mg/mL, respectively. The cells became rounded and dissymmetrical which reduced in number and size. The apoptotic cell death in ORL115 and ORL188 was deduced as caspase-3/7 activities that significantly increased (P < 0.05).
CONCLUSION: The study evidenced that the antioxidant activity of MTJ could influence the induction of apoptosis in ORL115 and ORL188 in future investigations and verifications.