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  1. Fulltext Tear Samples for Protein Extraction: Comparative Analysis of Schirmer's Test Strip and Microcapillary Tube Methods
    Tham ML, Mahmud A, Abdullah M, Md Saleh R, Mohammad Razali A, Cheah YK, et al.
    Cureus, 2023 Dec;15(12):e50972.
    PMID: 38259376 DOI: 10.7759/cureus.50972
    INTRODUCTION: Tear sampling is an attractive option for collecting biological samples in ophthalmology clinics, as it offers a non-invasive alternative to other invasive techniques. However, there are many tear sampling methods still in consideration. This study explores the suitability of Schirmer's test strip and microcapillary tube as reliable and satisfactory methods for tear sampling.

    METHODS: Tear samples were collected from eight healthy volunteers using the standard Schirmer's test strip method with or without anesthesia and microcapillary tubes. The total tear protein concentrations were analyzed via spectrophotometry and bicinchoninic acid (BCA) protein assay. The protein profile was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal wetting length of Schirmer's strip and suitable buffer solutions were compared. Discomfort levels reported by participants and the ease of execution for ophthalmologists were also evaluated.

    RESULTS: Tear samples exhibited typical protein profiles as shown by SDS-PAGE. The mean total protein obtained from an optimum wetting length of 20 mm using Schirmer's strip without anesthesia in phosphate-buffered saline (PBS) yielded substantial quantities of protein as measured by nanophotometer (220.20 ± 67.43 µg) and the BCA protein assay (210.34 ± 59.46 µg). This method collected a significantly higher quantity of protein compared to the microcapillary tube method (p=0.004) which was much more difficult to standardize. The clinician found it harder to utilize microcapillary tubes, while participants experienced higher insecurity and less discomfort with the microcapillary tube method. PBS used during the tear protein extraction process eluted higher tear protein concentration than ammonium bicarbonate, although the difference was not statistically significant. Using anaesthesia did not ease the sampling procedure substantially and protein quantity was maintained.

    CONCLUSION: Good quality and quantity of protein from tear samples were extracted with the optimized procedure. Schirmer's strip test in the absence of local anesthesia provided a standard, convenient, and non-invasive method for tear collection.

  2. Fulltext Structural and functional analyses of Burkholderia pseudomallei BPSL1038 reveal a Cas-2/VapD nuclease sub-family
    Shaibullah S, Shuhaimi N, Ker DS, Mohd-Sharif N, Ho KL, Teh AH, et al.
    Commun Biol, 2023 Sep 08;6(1):920.
    PMID: 37684342 DOI: 10.1038/s42003-023-05265-4
    Burkholderia pseudomallei is a highly versatile pathogen with ~25% of its genome annotated to encode hypothetical proteins. One such hypothetical protein, BPSL1038, is conserved across seven bacterial genera and 654 Burkholderia spp. Here, we present a 1.55 Å resolution crystal structure of BPSL1038. The overall structure folded into a modified βαββαβα ferredoxin fold similar to known Cas2 nucleases. The Cas2 equivalent catalytic aspartate (D11) pairs are conserved in BPSL1038 although B. pseudomallei has no known CRISPR associated system. Functional analysis revealed that BPSL1038 is a nuclease with endonuclease activity towards double-stranded DNA. The DNase activity is divalent ion independent and optimum at pH 6. The concentration of monovalent ions (Na+ and K+) is crucial for nuclease activity. An active site with a unique D11(X20)SST motif was identified and proposed for BPSL1038 and its orthologs. Structure modelling indicates the catalytic role of the D11(X20)SST motif and that the arginine residues R10 and R30 may interact with the nucleic acid backbone. The structural similarity of BPSL1038 to Cas2 proteins suggests that BPSL1038 may represent a sub-family of nucleases that share a common ancestor with Cas2.
  3. Fulltext Author Correction: Conditioned media of pancreatic cancer cells and pancreatic stellate cells induce myeloid-derived suppressor cells differentiation and lymphocytes suppression
    Chong YP, Peter EP, Lee FJM, Chan CM, Chai S, Ling LPC, et al.
    Sci Rep, 2023 Aug 10;13(1):13040.
    PMID: 37563191 DOI: 10.1038/s41598-023-40226-1
  4. Genomic analysis and biological characterization of a novel Schitoviridae phage infecting Vibrio alginolyticus
    Tajuddin S, Khan AM, Chong LC, Wong CL, Tan JS, Ina-Salwany MY, et al.
    Appl Microbiol Biotechnol, 2023 Feb;107(2-3):749-768.
    PMID: 36520169 DOI: 10.1007/s00253-022-12312-3
    Vibrio alginolyticus is a Gram-negative bacterium commonly associated with mackerel poisoning. A bacteriophage that specifically targets and lyses this bacterium could be employed as a biocontrol agent for treating the bacterial infection or improving the shelf-life of mackerel products. However, only a few well-characterized V. alginolyticus phages have been reported in the literature. In this study, a novel lytic phage, named ΦImVa-1, specifically infecting V. alginolyticus strain ATCC 17749, was isolated from Indian mackerel. The phage has a short latent period of 15 min and a burst size of approximately 66 particles per infected bacterium. ΦImVa-1 remained stable for 2 h at a wide temperature (27-75 °C) and within a pH range of 5 to 10. Transmission electron microscopy revealed that ΦImVa-1 has an icosahedral head of approximately 60 nm in diameter with a short tail, resembling those in the Schitoviridae family. High throughput sequencing and bioinformatics analysis elucidated that ΦImVa-1 has a linear dsDNA genome of 77,479 base pairs (bp), with a G + C content of ~ 38.72% and 110 predicted gene coding regions (106 open reading frames and four tRNAs). The genome contains an extremely large virion-associated RNA polymerase gene and two smaller non-virion-associated RNA polymerase genes, which are hallmarks of schitoviruses. No antibiotic genes were found in the ΦImVa-1 genome. This is the first paper describing the biological properties, morphology, and the complete genome of a V. alginolyticus-infecting schitovirus. When raw mackerel fish flesh slices were treated with ΦImVa-1, the pathogen loads reduced significantly, demonstrating the potential of the phage as a biocontrol agent for V. alginolyticus strain ATCC 17749 in the food. KEY POINTS: • A novel schitovirus infecting Vibrio alginolyticus ATCC 17749 was isolated from Indian mackerel. • The complete genome of the phage was determined, analyzed, and compared with other phages. • The phage is heat stable making it a potential biocontrol agent in extreme environments.
  5. Fulltext Conditioned media of pancreatic cancer cells and pancreatic stellate cells induce myeloid-derived suppressor cells differentiation and lymphocytes suppression
    Chong YP, Peter EP, Lee FJM, Chan CM, Chai S, Ling LPC, et al.
    Sci Rep, 2022 Jul 19;12(1):12315.
    PMID: 35853996 DOI: 10.1038/s41598-022-16671-9
    As pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs) are the two major cell types that comprise the immunosuppressive tumor microenvironment of pancreatic cancer, we aimed to investigate the role of conditioned medium derived from PCCs and PSCs co-culture on the viability of lymphocytes. The conditioned medium (CM) collected from PCCs and/or PSCs was used to treat peripheral blood mononuclear cells (PBMCs) to determine CM ability in reducing lymphocytes population. A proteomic analysis has been done on the CM to investigate the differentially expressed protein (DEP) expressed by two PCC lines established from different stages of tumor. Subsequently, we investigated if the reduction of lymphocytes was directly caused by CM or indirectly via CM-induced MDSCs. This was achieved by isolating lymphocyte subtypes and treating them with CM and CM-induced MDSCs. Both PCCs and PSCs were important in suppressing lymphocytes, and the PCCs derived from a metastatic tumor appeared to have a stronger suppressive effect than the PCCs derived from a primary tumor. According to the proteomic profiles of CM, 416 secreted proteins were detected, and 13 DEPs were identified between PANC10.05 and SW1990. However, CM was found unable to reduce lymphocytes viability through a direct pathway. In contrast, CM that contains proteins secreted by PCC and/or PSC appear immunogenic as they increase the viability of lymphocytes subtypes. Lymphocyte subtype treated with CM-induced MDSCs showed reduced viability in T helper 1 (Th1), T helper 2 (Th2), and T regulatory (Treg) cells, but not in CD8+ T cells, and B cells. As a conclusion, the interplay between PCCs and PSCs is important as their co-culture displays a different trend in lymphocytes suppression, hence, their co-culture should be included in future studies to better mimic the tumor microenvironment.
  6. Peperomia pellucida (L.) Kunth and eye diseases: A review on phytochemistry, pharmacology and toxicology
    Ho KL, Yong PH, Wang CW, Kuppusamy UR, Ngo CT, Massawe F, et al.
    J Integr Med, 2022 Feb 02.
    PMID: 35153134 DOI: 10.1016/j.joim.2022.02.002
    Peperomia pellucida (L.) Kunth is a medicinal plant used to manage inflammatory illnesses such as conjunctivitis, and gastrointestinal and respiratory tract disorders in tropical and subtropical regions. However, little is known about its pharmacological mechanism of action against eye diseases. This review aims to critically discuss the phytochemistry, pharmacology and toxicology of P. pellucida as well as its roles in the treatment of cataract, glaucoma and diabetic retinopathy. Recent developments in the uses of P. pellucida for healthcare and nutraceutical products by the pharmaceutical industry are also covered in this review. For this review, a literature search was performed with PubMed, ScienceDirect, SciFinder Scholar and Scopus databases, using relevant keywords. Among the various phytochemicals identified from P. pellucida, β-caryophyllene, carotol, dillapiole, ellagic acid, pellucidin A, phytol and vitexin exhibit strong pharmacological activities within the mitogen-activated protein kinase and nuclear factor-κB signalling pathways in inflammatory eye diseases. The antihypertensive, anti-inflammatory, antioxidant, antihyperglycemic and anti-angiogenic activities displayed by P. pellucida extracts in many in vitro, in vivo and clinical studies suggest its potential role in the management of inflammatory eye diseases. P. pellucida extract was non-toxic against normal cell lines but displayed mild toxicity in animal models. The growing public interest in P. pellucida has inspired the nutraceutical and pharmaceutical industries to process the plant into health products. Although the potential pharmacological mechanisms against eye diseases have been summarized, further studies of the interactions among constituent phytochemicals from P. pellucida within various signalling pathways shall support the use of the plant as an alternative therapeutic source.
  7. Fulltext Loop-Mediated Isothermal Amplification (LAMP) as a Promising Point-of-Care Diagnostic Strategy in Avian Virus Research
    Padzil F, Mariatulqabtiah AR, Tan WS, Ho KL, Isa NM, Lau HY, et al.
    Animals (Basel), 2021 Dec 30;12(1).
    PMID: 35011181 DOI: 10.3390/ani12010076
    Over the years, development of molecular diagnostics has evolved significantly in the detection of pathogens within humans and their surroundings. Researchers have discovered new species and strains of viruses, while mitigating the viral infections that occur, owing to the accessibility of nucleic acid screening methods such as polymerase chain reaction (PCR), quantitative (real-time) polymerase chain reaction (qPCR) and reverse-transcription qPCR (RT-qPCR). While such molecular detection methods are widely utilized as the benchmark, the invention of isothermal amplifications has also emerged as a reliable tool to improvise on-field diagnosis without dependence on thermocyclers. Among the established isothermal amplification technologies are loop-mediated isothermal amplification (LAMP), recombinant polymerase amplification (RPA), strand displacement activity (SDA), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA) and rolling circle amplification (RCA). This review highlights the past research on and future prospects of LAMP, its principles and applications as a promising point-of-care diagnostic method against avian viruses.
  8. Fulltext Immunological Analysis of Nodavirus Capsid Displaying the Domain III of Japanese Encephalitis Virus Envelope Protein
    Kumar K, Ong HK, Tan WS, Arshad SS, Ho KL
    Pharmaceutics, 2021 Nov 01;13(11).
    PMID: 34834244 DOI: 10.3390/pharmaceutics13111826
    Japanese encephalitis virus (JEV) is the pathogen that causes Japanese encephalitis (JE) in humans and horses. Lethality of the virus was reported to be between 20-30%, of which, 30-50% of the JE survivors develop neurological and psychiatric sequelae. Attributed to the low effectiveness of current therapeutic approaches against JEV, vaccination remains the only effective approach to prevent the viral infection. Currently, live-attenuated and chimeric-live vaccines are widely used worldwide but these vaccines pose a risk of virulence restoration. Therefore, continuing development of JE vaccines with higher safety profiles and better protective efficacies is urgently needed. In this study, the Macrobrachium rosenbergii nodavirus (MrNV) capsid protein (CP) fused with the domain III of JEV envelope protein (JEV-DIII) was produced in Escherichia coli. The fusion protein (MrNV-CPJEV-DIII) assembled into virus-like particles (VLPs) with a diameter of approximately 18 nm. The BALB/c mice injected with the VLPs alone or in the presence of alum successfully elicited the production of anti-JEV-DIII antibody, with titers significantly higher than that in mice immunized with IMOJEV, a commercially available vaccine. Immunophenotyping showed that the MrNV-CPJEV-DIII supplemented with alum triggered proliferation of cytotoxic T-lymphocytes, macrophages, and natural killer (NK) cells. Additionally, cytokine profiles of the immunized mice revealed activities of cytotoxic T-lymphocytes, macrophages, and NK cells, indicating the activation of adaptive cellular and innate immune responses mediated by MrNV-CPJEV-DIII VLPs. Induction of innate, humoral, and cellular immune responses by the MrNV-CPJEV-DIII VLPs suggest that the chimeric protein is a promising JEV vaccine candidate.
  9. Microarray-based identification of differentially expressed genes associated with andrographolide derivatives-induced resistance in colon and prostate cancer cell lines
    Quah SY, Wong CC, Wong HC, Ho KL, Abdul Manan N, Deb PK, et al.
    Toxicol Appl Pharmacol, 2021 08 15;425:115605.
    PMID: 34087331 DOI: 10.1016/j.taap.2021.115605
    Chemoresistance poses a major hurdle to cancer treatments. Andrographolide-derived SRJ09 and SRJ23 were reported to exhibit potent, selective inhibitory activities against colon and prostate cancer cells, respectively. In this study, previously developed resistant colon (HCT-116rst09) and prostate (PC-3rst23) cancer cell lines were used to elucidate the molecular mechanisms contributing to chemoresistance. Cytotoxic effects of SRJ09 and SRJ23 on both parental and resistant cells were investigated. Cell cycle distributions in HCT-116rst09 cells following SRJ09 treatment were analysed using flow cytometry. Whole-genome microarray analysis was performed on both parental and resistant cells to obtain differential gene expression profiles. Microarray data were subjected to protein-protein interaction network, functional enrichment, and pathway analyses. Reverse transcription-polymerase chain reaction (RT-PCR) was used to validate the changes in expression levels of selected genes. Besides morphological changes, HCT-116rst09 cells showed 7.0-fold resistance to SRJ09 while PC-3rst23 cells displayed a 5.5-fold resistance to SRJ23, as compared with their respective parental cells. G0/G1-phase cell cycle arrest was observed in HCT-116rst09 cells upon SRJ09 treatment. Collectively, 77 and 21 genes were found differentially modulated in HCT-116rst09 and PC-3rst23 cells, respectively. Subsequent bioinformatics analysis revealed several genes associated with FGFR4 and PI3K pathways, and cancer stemness, were chemoresistance mediators in HCT-116rst09 cells. RT-PCR confirmed the HMOX1 upregulation and ATG12 downregulation protected the PC-3rst23 cells from SRJ23 cytotoxicity. In conclusion, acquired chemoresistance to SRJ09 and SRJ23 in colon and prostate cancer cells, respectively, could be attributed to the alterations in the expression of genes such as those related to PI3K and autophagy pathways.
  10. Fulltext Regulation of Proteolytic Activity to Improve the Recovery of Macrobrachium rosenbergii Nodavirus Capsid Protein
    Selvaraj BA, Mariatulqabtiah AR, Ho KL, Ng CL, Yong CY, Tan WS
    Int J Mol Sci, 2021 Aug 13;22(16).
    PMID: 34445426 DOI: 10.3390/ijms22168725
    The causative agent of white tail disease (WTD) in the giant freshwater prawn is Macrobrachium rosenbergii nodavirus (MrNV). The recombinant capsid protein (CP) of MrNV was previously expressed in Escherichia coli, and it self-assembled into icosahedral virus-like particles (VLPs) with a diameter of approximately 30 nm. Extensive studies on the MrNV CP VLPs have attracted widespread attention in their potential applications as biological nano-containers for targeted drug delivery and antigen display scaffolds for vaccine developments. Despite their advantageous features, the recombinant MrNV CP VLPs produced in E. coli are seriously affected by protease degradations, which significantly affect the yield and stability of the VLPs. Therefore, the aim of this study is to enhance the stability of MrNV CP by modulating the protease degradation activity. Edman degradation amino acid sequencing revealed that the proteolytic cleavage occurred at arginine 26 of the MrNV CP. The potential proteases responsible for the degradation were predicted in silico using the Peptidecutter, Expasy. To circumvent proteolysis, specific protease inhibitors (PMSF, AEBSF and E-64) were tested to reduce the degradation rates. Modulation of proteolytic activity demonstrated that a cysteine protease was responsible for the MrNV CP degradation. The addition of E-64, a cysteine protease inhibitor, remarkably improved the yield of MrNV CP by 2.3-fold compared to the control. This innovative approach generates an economical method to improve the scalability of MrNV CP VLPs using individual protease inhibitors, enabling the protein to retain their structural integrity and stability for prominent downstream applications including drug delivery and vaccine development.
  11. Fulltext Hepatitis B Virus-Like Particle: Targeted Delivery of Plasmid Expressing Short Hairpin RNA for Silencing the Bcl-2 Gene in Cervical Cancer Cells
    Akwiditya MA, Yong CY, Yusof MT, Mariatulqabtiah AR, Ho KL, Tan WS
    Int J Mol Sci, 2021 Feb 26;22(5).
    PMID: 33652577 DOI: 10.3390/ijms22052320
    Gene therapy research has advanced to clinical trials, but it is hampered by unstable nucleic acids packaged inside carriers and there is a lack of specificity towards targeted sites in the body. This study aims to address gene therapy limitations by encapsidating a plasmid synthesizing a short hairpin RNA (shRNA) that targets the anti-apoptotic Bcl-2 gene using truncated hepatitis B core antigen (tHBcAg) virus-like particle (VLP). A shRNA sequence targeting anti-apoptotic Bcl-2 was synthesized and cloned into the pSilencer 2.0-U6 vector. The recombinant plasmid, namely PshRNA, was encapsidated inside tHBcAg VLP and conjugated with folic acid (FA) to produce FA-tHBcAg-PshRNA VLP. Electron microscopy revealed that the FA-tHBcAg-PshRNA VLP has an icosahedral structure that is similar to the unmodified tHBcAg VLP. Delivery of FA-tHBcAg-PshRNA VLP into HeLa cells overexpressing the folate receptor significantly downregulated the expression of anti-apoptotic Bcl-2 at 48 and 72 h post-transfection. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay demonstrated that the cells' viability was significantly reduced from 89.46% at 24 h to 64.52% and 60.63%, respectively, at 48 and 72 h post-transfection. As a conclusion, tHBcAg VLP can be used as a carrier for a receptor-mediated targeted delivery of a therapeutic plasmid encoding shRNA for gene silencing in cancer cells.
  12. Fulltext Chimeric Virus-Like Particles of Prawn Nodavirus Displaying Hepatitis B Virus Immunodominant Region: Biophysical Properties and Cytokine Response
    Ninyio NN, Ho KL, Yong CY, Chee HY, Hamid M, Ong HK, et al.
    Int J Mol Sci, 2021 Feb 15;22(4).
    PMID: 33672018 DOI: 10.3390/ijms22041922
    Hepatitis B is a major global health challenge. In the absence of an effective treatment for the disease, hepatitis B vaccines provide protection against the viral infection. However, some individuals do not have positive immune responses after being vaccinated with the hepatitis B vaccines available in the market. Thus, it is important to develop a more protective vaccine. Previously, we showed that hepatitis B virus (HBV) 'a' determinant (aD) displayed on the prawn nodavirus capsid (Nc) and expressed in Spodoptera frugiperda (Sf9) cells (namely, Nc-aD-Sf9) self-assembled into virus-like particles (VLPs). Immunisation of BALB/c mice with the Nc-aD-Sf9 VLPs showed significant induction of humoral, cellular and memory B-cell immunity. In the present study, the biophysical properties of the Nc-aD-Sf9 VLPs were studied using dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy. Enzyme-linked immunosorbent assay (ELISA) was used to determine the antigenicity of the Nc-aD-Sf9 VLPs, and multiplex ELISA was employed to quantify the cytokine response induced by the VLPs administered intramuscularly into BALB/c mice (n = 8). CD spectroscopy of Nc-aD-Sf9 VLPs showed that the secondary structure of the VLPs predominantly consisted of beta (β)-sheets (44.8%), and they were thermally stable up to ~52 °C. ELISA revealed that the aD epitope of the VLPs was significantly antigenic to anti-HBV surface antigen (HBsAg) antibodies. In addition, multiplex ELISA of serum samples from the vaccinated mice showed a significant induction (p < 0.001) of IFN-γ, IL-4, IL-5, IL-6, IL-10, and IL-12p70. This cytokine profile is indicative of natural killer cell, macrophage, dendritic cell and cytotoxic T-lymphocyte activities, which suggests a prophylactic innate and adaptive cellular immune response mediated by Nc-aD-Sf9 VLPs. Interestingly, Nc-aD-Sf9 induced a more robust release of the aforementioned cytokines than that of Nc-aD VLPs produced in Escherichia coli and a commercially used hepatitis B vaccine. Overall, Nc-aD-Sf9 VLPs are thermally stable and significantly antigenic, demonstrating their potential as an HBV vaccine candidate.
  13. Fulltext Molecular detection and characterisation of Domestic Cat Hepadnavirus (DCH) from blood and liver tissues of cats in Malaysia
    Anpuanandam K, Selvarajah GT, Choy MMK, Ng SW, Kumar K, Ali RM, et al.
    BMC Vet Res, 2021 Jan 06;17(1):9.
    PMID: 33407487 DOI: 10.1186/s12917-020-02700-0
    BACKGROUND: A new domestic cat hepadnavirus (DCH, family Hepadnaviridae) was first reported from whole blood samples of domestic cats in Australia in 2018, and from cat serum samples in Italy in 2019. The pathogenesis of DCH is unknown, but it was reported in cats with viraemia (6.5-10.8%), chronic hepatitis (43%) and hepatocellular carcinoma (28%). Recent reports suggest that DCH resembles the human hepatitis B virus (HBV) and its related hepatopathies. This study aims to detect and characterize DCH among domestic cats in Malaysia. A cross-sectional study was performed on 253 cats, of which 87 had paired blood and liver samples, entailing whole-genome sequencing and phylogenetic analysis of DCH from a liver tissue sample.

    RESULTS: Among the 253 cats included in this study, 12.3% of the whole blood samples tested positive for DCH. The detection rate was significantly higher in pet cats (16.6%, n = 24/145) compared to shelter cats (6.5%, n = 7/108). Liver tissues showed higher a DCH detection rate (14.9%, n = 13/87) compared to blood; 5 out of these 13 cats tested positive for DCH in their paired liver and blood samples. Serum alanine transaminase (ALT) was elevated (> 95 units/L) in 12 out of the 23 DCH-positive cats (52.2%, p = 0.012). Whole-genome sequence analysis revealed that the Malaysian DCH strain, with a genome size of 3184 bp, had 98.3% and 97.5% nucleotide identities to the Australian and Italian strains, respectively. The phylogenetic analysis demonstrated that the Malaysian DCH genome was clustered closely to the Australian strain, suggesting that they belong to the same geographically-determined genetic pool (Australasia).

    CONCLUSIONS: This study provided insights into a Malaysian DCH strain that was detected from a liver tissue. Interestingly, pet cats or cats with elevated ALT were significantly more likely to be DCH positive. Cats with positive DCH detection from liver tissues may not necessarily have viraemia. The impact of this virus on inducing liver diseases in felines warrants further investigation.

  14. Fulltext Transcriptomic and proteomic profiling revealed reprogramming of carbon metabolism in acetate-grown human pathogen Candida glabrata
    Chew SY, Brown AJP, Lau BYC, Cheah YK, Ho KL, Sandai D, et al.
    J Biomed Sci, 2021 Jan 02;28(1):1.
    PMID: 33388061 DOI: 10.1186/s12929-020-00700-8
    BACKGROUND: Emergence of Candida glabrata, which causes potential life-threatening invasive candidiasis, has been widely associated with high morbidity and mortality. In order to cause disease in vivo, a robust and highly efficient metabolic adaptation is crucial for the survival of this fungal pathogen in human host. In fact, reprogramming of the carbon metabolism is believed to be indispensable for phagocytosed C. glabrata within glucose deprivation condition during infection.

    METHODS: In this study, the metabolic responses of C. glabrata under acetate growth condition was explored using high-throughput transcriptomic and proteomic approaches.

    RESULTS: Collectively, a total of 1482 transcripts (26.96%) and 242 proteins (24.69%) were significantly up- or down-regulated. Both transcriptome and proteome data revealed that the regulation of alternative carbon metabolism in C. glabrata resembled other fungal pathogens such as Candida albicans and Cryptococcus neoformans, with up-regulation of many proteins and transcripts from the glyoxylate cycle and gluconeogenesis, namely isocitrate lyase (ICL1), malate synthase (MLS1), phosphoenolpyruvate carboxykinase (PCK1) and fructose 1,6-biphosphatase (FBP1). In the absence of glucose, C. glabrata shifted its metabolism from glucose catabolism to anabolism of glucose intermediates from the available carbon source. This observation essentially suggests that the glyoxylate cycle and gluconeogenesis are potentially critical for the survival of phagocytosed C. glabrata within the glucose-deficient macrophages.

    CONCLUSION: Here, we presented the first global metabolic responses of C. glabrata to alternative carbon source using transcriptomic and proteomic approaches. These findings implicated that reprogramming of the alternative carbon metabolism during glucose deprivation could enhance the survival and persistence of C. glabrata within the host.

  15. Fulltext Virus-like Particle Vaccines: A Prospective Panacea Against an Avian Influenza Panzootic
    Ninyio NN, Ho KL, Omar AR, Tan WS, Iqbal M, Mariatulqabtiah AR
    Vaccines (Basel), 2020 Nov 19;8(4).
    PMID: 33227887 DOI: 10.3390/vaccines8040694
    Epizootics of highly pathogenic avian influenza (HPAI) have resulted in the deaths of millions of birds leading to huge financial losses to the poultry industry worldwide. The roles of migratory wild birds in the harbouring, mutation, and transmission of avian influenza viruses (AIVs), and the lack of broad-spectrum prophylactic vaccines present imminent threats of a global panzootic. To prevent this, control measures that include effective AIV surveillance programmes, treatment regimens, and universal vaccines are being developed and analysed for their effectiveness. We reviewed the epidemiology of AIVs with regards to past avian influenza (AI) outbreaks in birds. The AIV surveillance programmes in wild and domestic birds, as well as their roles in AI control were also evaluated. We discussed the limitations of the currently used AI vaccines, which necessitated the development of a universal vaccine. We evaluated the current development of AI vaccines based upon virus-like particles (VLPs), particularly those displaying the matrix-2 ectodomain (M2e) peptide. Finally, we highlighted the prospects of these VLP vaccines as universal vaccines with the potential of preventing an AI panzootic.
  16. Fulltext Targeted delivery of 5-fluorouracil-1-acetic acid (5-FA) to cancer cells overexpressing epithelial growth factor receptor (EGFR) using virus-like nanoparticles
    Gan BK, Rullah K, Yong CY, Ho KL, Omar AR, Alitheen NB, et al.
    Sci Rep, 2020 Oct 08;10(1):16867.
    PMID: 33033330 DOI: 10.1038/s41598-020-73967-4
    Chemotherapy is widely used in cancer treatments. However, non-specific distribution of chemotherapeutic agents to healthy tissues and normal cells in the human body always leads to adverse side effects and disappointing therapeutic outcomes. Therefore, the main aim of this study was to develop a targeted drug delivery system based on the hepatitis B virus-like nanoparticle (VLNP) for specific delivery of 5-fluorouracil-1-acetic acid (5-FA) to cancer cells expressing epithelial growth factor receptor (EGFR). 5-FA was synthesized from 5-fluorouracil (5-FU), and it was found to be less toxic than the latter in cancer cells expressing different levels of EGFR. The cytotoxicity of 5-FA increased significantly after being conjugated on the VLNP. A cell penetrating peptide (CPP) of EGFR was displayed on the VLNP via the nanoglue concept, for targeted delivery of 5-FA to A431, HT29 and HeLa cells. The results showed that the VLNP displaying the CPP and harboring 5-FA internalized the cancer cells and killed them in an EGFR-dependent manner. This study demonstrated that the VLNP can be used to deliver chemically modified 5-FU derivatives to cancer cells overexpressing EGFR, expanding the applications of the VLNP in targeted delivery of chemotherapeutic agents to cancer cells overexpressing this transmembrane receptor.
  17. In silico and saturation transfer difference NMR approaches to unravel the binding mode of an andrographolide derivative to K-Ras oncoprotein
    Quah SY, Tan MS, Ho KL, Manan NA, Gorfe AA, Deb PK, et al.
    Future Med Chem, 2020 09;12(18):1611-1631.
    PMID: 32892640 DOI: 10.4155/fmc-2020-0104
    Background: Andrographolide and its benzylidene derivatives, SRJ09 and SRJ23, potentially bind oncogenic K-Ras to exert anticancer activity. Their molecular interactions with K-Ras oncoproteins that lead to effective biological activity are of major interest. Methods & results: In silico docking and molecular dynamics simulation were performed using Glide and Desmond, respectively; while saturation transfer difference NMR was performed using GDP-bound K-RasG12V. SRJ23 was found to bind strongly and selectively to K-RasG12V, by anchoring to a binding pocket (namely p2) principally via hydrogen bond and hydrophobic interactions. The saturation transfer difference NMR analysis revealed the proximity of protons of functional moieties in SRJ23 to K-RasG12V, suggesting positive binding. Conclusion: SRJ23 binds strongly and interacts stably with K-RasG12V to exhibit its inhibitory activity.
  18. Fulltext Immunological Analysis of the Hepatitis B Virus "a" Determinant Displayed on Chimeric Virus-Like Particles of Macrobrachium rosenbergii Nodavirus Capsid Protein Produced in Sf9 Cells
    Ninyio NN, Ho KL, Ong HK, Yong CY, Chee HY, Hamid M, et al.
    Vaccines (Basel), 2020 Jun 04;8(2).
    PMID: 32512923 DOI: 10.3390/vaccines8020275
    Chimeric virus-like particles (VLPs) have been widely exploited for various purposes including their use as vaccine candidates, particularly due to their ability to induce stronger immune responses than VLPs consisting of single viral proteins. In the present study, VLPs of the Macrobrachium rosenbergii nodavirus (MrNV) capsid protein (Nc) displaying the hepatitis B virus "a" determinant (aD) were produced in Spodoptera frugiperda (Sf9) insect cells. BALB/c mice immunised with the purified chimeric Nc-aD VLPs elicited a sustained titre of anti-aD antibody, which was significantly higher than that elicited by a commercially available hepatitis B vaccine and Escherichia coli-produced Nc-aD VLPs. Immunophenotyping showed that the Sf9-produced Nc-aD VLPs induced proliferation of cytotoxic T-lymphocytes and NK1.1 natural killer cells. Furthermore, enzyme-linked immunospot (ELISPOT)analysis showed the presence of antibody-secreting memory B cells in the mice splenocytes stimulated with the synthetic aD peptide. The significant humoral, natural killer cell and memory B cell immune responses induced by the Sf9-produced Nc-aD VLPs suggest that they present good prospects for use as a hepatitis B vaccine candidate.
  19. An Application of pET SUMO Protein Expression System in Escherichia coli: Cloning, Expression, Purification, and Characterisation of Native Kras4BG12V Oncoprotein
    Tan MS, Teh YH, Ho KL, Stanslas J
    Protein J, 2020 02;39(1):54-61.
    PMID: 31620959 DOI: 10.1007/s10930-019-09872-1
    Being an important regulator of cell growth and survival, a point mutation at glycine-12 residue of Kras4B to valine (V), renders Kras4BG12V oncogenic. Kras4B recombinant protein is used as a bait to fish its potential ligands in the attempt of drugging this oncoprotein and to validate its pharmacologically relevant ligand in protein-ligand interaction studies. Nevertheless, synthesis of Kras4B recombinant protein is challenging as it was reported being susceptible to aggregation into inclusion bodies in the bacterial host, resulting in a poor yield of recombinant protein. Here, we describe a novel method to produce native Kras4BG12V protein by using pET SUMO protein expression system as a solution to the formation of inclusion bodies. Kras4BG12V oncogene was cloned into pET SUMO vector, followed by a 12 h chemically induced protein expression in Escherichia coli at 20 °C. Native Kras4BG12V protein was produced in a series of protein purification steps involving immobilised nickel ion-affinity column chromatography, SUMO fusion protein and polyhistidine tag removal, and size exclusion column chromatography. The identity of the purified Kras4BG12V protein was validated by immunoblot analysis. The purified protein exhibited self-dimerising, indicating that the purified protein structurally resembles Kras4B. Its physical interaction with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a known binder of Kras4B, confirms the identity of the purified protein as Kras4BG12V. The native Kras4BG12V protein was successfully purified in a substantial amount by using the pET SUMO protein expression system.
  20. Fulltext Advances in the Diagnosis of Foot-and-Mouth Disease
    Wong CL, Yong CY, Ong HK, Ho KL, Tan WS
    Front Vet Sci, 2020;7:477.
    PMID: 32974392 DOI: 10.3389/fvets.2020.00477
    Foot-and-mouth disease (FMD) is a devastating livestock disease caused by foot-and-mouth disease virus (FMDV). Outbreaks of this disease in a country always result in conspicuous economic losses to livestock industry and subsequently lead to serious socioeconomic damages due to the immediate imposition of trade embargo. Rapid and accurate diagnoses are imperative to control this infectious virus. In the current review, enzyme-linked immunosorbent assay (ELISA)-based methods used in FMD diagnosis are extensively reviewed, particularly the sandwich, liquid-phase blocking, and solid-phase competition ELISA. The differentiation of infected animals from vaccinated animals using ELISA-based methods is also highlighted, in which the role of 3ABC polyprotein as a marker is reviewed intensively. Recently, more studies are focusing on the molecular diagnostic methods, which detect the viral nucleic acids based on reverse transcription-polymerase chain reaction (RT-PCR) and RT-loop-mediated isothermal amplification (RT-LAMP). These methods are generally more sensitive because of their ability to amplify a minute amount of the viral nucleic acids. In this digital era, the RT-PCR and RT-LAMP are progressing toward the mobile versions, aiming for on-site FMDV diagnosis. Apart from RT-PCR and RT-LAMP, another diagnostic assay specifically designed for on-site diagnosis is the lateral flow immunochromatographic test strips. These test strips have some distinct advantages over other diagnostic methods, whereby the assay often does not require the aid of an external device, which greatly lowers the cost per test. In addition, the on-site diagnostic test can be easily performed by untrained personnel including farmers, and the results can be obtained in a few minutes. Lastly, the use of FMDV diagnostic assays for progressive control of the disease is also discussed critically.
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