Aims: The kelch13 gene mutations of Plasmodium falciparum is associated with delayed parasite clearance after artemisinin-based combination therapy (ACT). It is unclear for P. knowlesi that is predominantly reported in Sabah. Therefore, this study aims to analyse the diversity of the P. knowlesi kelch13 gene in five divisions of Sabah. Methodology and results: : Ninety-five blood samples infected with P. knowlesi were obtained. The DNA of P. knowlesi samples was extracted and the kelch13 gene was amplified. The amplicons were cloned and sequenced. The sequencing data were aligned and analysed using MEGA 11 and DnaSP v6 software. A phylogenetic tree was constructed using the Neighbour-joining approach, which showed a diverse clade of P. knowlesi in Sabah, with a nucleotide diversity (π) of 0.451 and a haplotype diversity of 0.947. The deduced amino acid sequences were classified into 14 haplotypes, providing evidence of distinct P. knowlesi lineages in Sabah. When compared to P. falciparum, the kelch13 sequences of P. knowlesi exhibited a higher π of 0.490 and haplotype diversity of 1.000, and similar mutations that conferred drug resistance to ACT in P. falciparum were detected in P. knowlesi in this study. Conclusion, significance and impact of study: The kelch13 gene of P. knowlesi isolates in Sabah has high nucleotide and haplotype diversities. Additionally, mutations conferring drug resistance to ACT in P. falciparum were identified in P. knowlesi in Sabah. The findings in this study can be used to better understand the emergence of drug resistance of P. knowlesi in Sabah.

Aims: Glycogen synthase kinase-3 (GSK-3 (EC:2.7.11.1)) is one of the main therapeutic targets for treating cancer, diabetes, neurological illness and parasitic infection. Due to their distinctive structural characteristics and wide-ranging biological actions, small compounds from soil bacteria have been the most sought-after source for GSK-3 inhibitors. This study assessed the activities of soil actinomycetes isolated from Sabah, Malaysia, against human GSK-3β. Methodology and results: A total of 514 actinomycetes strains were isolated from 144 soil samples. The activities of the crude extracts were evaluated against GSK-3β and its upstream regulators (MKK1 and PP1/GLC7) using yeast-based assays. Eight actinomycetes extracts showed selective human GSK-3β inhibition without affecting MKK1 and PP1/GLC7. The extract from one of these eight isolates, FA013, also showed potent and selective anti-plasmodial activities against Plasmodium falciparum 3D7 strain (IC50 = 0.18 μg/mL, SI = 13,850) with a non-toxic effect against Chang liver cells. Conclusion, significance and impact of study This study identified FA013 as a potential isolate from Malaysian rainforest soil with inhibitory activities against GSK-3β and malaria parasites for future drug development.

Aims: Calmodulin (CaM) is vital for the survival of Plasmodium knowlesi, a simian malaria parasite that infects both macaques and humans in Southeast Asia. To advance antimalarial drugs development targeting this protein, it is imperative to produce ample quantities of pure CaM for further research. Hence, this study aims to establish a robust strategy for the heterologous expression and purification of CaM from Plasmodium knowlesi (Pk-CaM). Methodology and results: First, we optimised the gene sequence of Pk-CaM for Escherichia coli expression, chemically synthesised it and integrated it into the pET28a plasmid. The optimised gene displayed a 45.15% GC content and a 0.81 codon-adaptation index, making it highly compatible with E. coli. Pk-CaM expression was assessed under various conditions, with the best results achieved at a post-induction temperature of 20 °C for 16 h, yielding a fully soluble protein. Subsequently, we purified the protein using Ni2+-NTA affinity chromatography and size-exclusion chromatography (SEC), obtaining 15 mg from 1 L of culture. The folding properties of purified Pk-CaM were analysed using far-UV circular dichroism (CD) spectroscopy, revealing a predominance of helical structures, both with and without Ca2+ ions. Binding to Ca2+ ions induced structural changes, increasing the helical content compared to when Ca2+ ions were absent. Conclusion, significance and impact of study The optimal conditions for the recombinant expression and purification of Pk-CaM in a correctly folded and functional form were successfully established in this study. This achievement provides a solid foundation for conducting further comprehensive research in the pursuit of novel antimalarial drugs.

Aim: FK506-binding protein 35 from Plasmodium knowlesi (Pk-FKBP35), a member of peptidyl-prolyl cis-trans isomerase (PPIase), is considered a viable target for the development of the novel antimalarial drug targeting zoonotic malaria in Malaysia. While FK506 effectively inhibits this protein, this drug is not applicable due to its immunosuppressive effects. This study aims to assess the inhibitory potential of different collagen hydrolysates (CH) against Pk-FKBP35, as FK506 replacers. Methodology and results: Recombinant full-length Pk-FKBP35 was initially over-expressed using Escherichia coli (BL21) host cells and subsequently purified via affinity chromatography coupled with size-exclusion chromatography. In this study, four distinct CH were employed, originating from bovine, bone broth, fish and swine. The results revealed that all CH inhibited PPIase catalytic activity of Pk-FKBP35 with IC50 values 1.63 mg/mL (bovine CH), 2.97 mg/mL (fish CH), 33.01 mg/mL (swine CH) and 13.91 mg/mL (bone broth CH), which were much higher than that of FK506. Furthermore, these CHs retained the ability of Pk-FKBP35 to inhibit calcineurin phosphatase activity, yet not as extreme as FK506. Conclusion, significance and impact of study The inhibition is predicted due to the presence of proline-rich peptides in CH, which were able to block the substrate binding cavity of Pk-FKBP35. This study suggested that CH might have no serious immunosuppressant effect and is promising for further harnessing for antimalarial compounds

Contrast Media ; Gadolinium ; Glomerular Filtration Rate ; Humans ; Magnetic Resonance Imaging ; Mass Screening

Aims: Streptococcus pneumoniae is one the world’s foremost bacterial pathogens that cause massive global mortality and morbidity in young children and immunocompromised adults especially in developing countries. Biofilms have been increasingly recognized as an important prerequisite to disease. Individual S. pneumoniae strains differ markedly in their virulence phenotypes, but genetic heterogeneity has complicated attempts to identify any association between a given clonal lineage and propensity to cause a particular disease type. This study investigated serotype 19 S. pneumoniae from blood and ear isolates for biofilm formation capacity in relation to isolate source, pH and ferric oxide [Fe(III)] supplementation. Methodology and results: Viable count and density biofilm assays, microscopy and multi locus sequence typing (MLST) were applied to investigate biofilm formation capacity and genetic diversity of serotype 19 S. pneumoniae from blood and ear isolates. Generally, blood isolates were observed to produce more biofilms at both pH 7.4 and 6.8 compared to the ear isolates. The supplementation of Fe(III) was also found to support biofilm growth. Upon MLST typing of the isolates, marked differences in biofilm formation within the same sequence types (ST) of ST199 and ST177 was observed. This strongly indicated that strains within the same sequence type show differences in biofilm formation capacity. Conclusion, significance and impact of study This study showed that despite belonging to the same serotype, serotype 19, S. pneumoniae blood and ear isolates showed high diversity in biofilm formation ability in relation to pH and Fe(III) supplementation. Additionally, pneumococcal isolates from sequence types ST199 and ST177 also gave rise to differences in biofilm formation ability within the same sequence type (ST). The diversity of biofilm formation within serotype 19 seen in this study is significant to further inform of vaccination strategies against pneumococcal infections, in that due to variations in biofilm formation capacity within the same ST. It is possible that within serotype 19 may show variable vaccination or drug treatment responses. This also indicates that the current treatment strategy which employs specific serotype selection as for PCV14 and PCV7 pneumococcal vaccines may not produce the desired therapeutic results.
Streptococcus pneumoniae--immunology ; Biofilms--radiation effects

The Singapore Health Services cluster (SingHealth) radiology film archives are a valuable repository of local radiological cases dating back to the 1950s. Some of the cases in the archives are of historical medical interest, i.e. cerebral angiography in the workup of patients with hemiplegia. Other cases are of historical social interest, being conditions seen during earlier stages of Singapore's development, i.e. bound feet. The archives form a unique portal into the development of local radiology as well as the national development of Singapore. A selection from the archives is published in commemoration of the International Day of Radiology in 2020, as well as the 200th anniversary of the Singapore General Hospital in 2021. This pictorial essay comprises gastroenterology, musculoskeletal and obstetrics and gynaecology cases from the archives.

BACKGROUND: The prevalence of peanut allergy (PA) among children has increased significantly over the past decade. Even though the prevalence of PA in Singapore is considered low, peanut is the top trigger for food-induced anaphylaxis in Singaporean children.OBJECTIVE: To describe the demographic characteristics and clinical features of children with PA.METHODS: This is a 5-year retrospective review of children diagnosed with PA based on clinical history coupled with a positive skin prick test to peanut or positive oral food challenge results.RESULTS: There were 269 patients (53.9% males) with a clinical diagnosis of PA. The median age at first allergic presentation for the PA group was 24 months old, with interquartile range of 13–39 months. The most common form of peanut introduced was roasted peanut. The rate of peanut anaphylaxis was 7.1%. Concomitant tree nut sensitization was found in 32.3% of this cohort, predominantly to cashew nut. Majority of them have a personal history of atopy – 75.8% with eczema, 63.6% with allergic rhinitis, and 19.7% with asthma.CONCLUSION: This is the first large review of peanut-allergic children in Singapore. Prospective population-based studies are needed to establish the true prevalence and risk factors associated with the development of this potentially life-threatening condition.
Anacardium ; Anaphylaxis ; Arachis ; Asia ; Asthma ; Child ; Cohort Studies ; Diagnosis ; Eczema ; Humans ; Nuts ; Peanut Hypersensitivity ; Prevalence ; Prospective Studies ; Retrospective Studies ; Rhinitis, Allergic ; Risk Factors ; Singapore ; Skin ; Trees