Displaying publications 41 - 59 of 59 in total

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  1. Chai KP, Othman NF, Teh AH, Ho KL, Chan KG, Shamsir MS, et al.
    Sci Rep, 2016 Mar 15;6:23126.
    PMID: 26975884 DOI: 10.1038/srep23126
    A new subfamily of glycosyl hydrolase family GH13 was recently proposed for α-amylases from Anoxybacillus species (ASKA and ADTA), Geobacillus thermoleovorans (GTA, Pizzo, and GtamyII), Bacillus aquimaris (BaqA), and 95 other putative protein homologues. To understand this new GH13 subfamily, we report crystal structures of truncated ASKA (TASKA). ASKA is a thermostable enzyme capable of producing high levels of maltose. Unlike GTA, biochemical analysis showed that Ca(2+) ion supplementation enhances the catalytic activities of ASKA and TASKA. The crystal structures reveal the presence of four Ca(2+) ion binding sites, with three of these binding sites are highly conserved among Anoxybacillus α-amylases. This work provides structural insights into this new GH13 subfamily both in the apo form and in complex with maltose. Furthermore, structural comparison of TASKA and GTA provides an overview of the conformational changes accompanying maltose binding at each subsite.
  2. Rahaman SN, Mat Yusop J, Mohamed-Hussein ZA, Ho KL, Teh AH, Waterman J, et al.
    Acta Crystallogr F Struct Biol Commun, 2016 Mar;72(Pt 3):207-13.
    PMID: 26919524 DOI: 10.1107/S2053230X16002016
    C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.
  3. 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.
  4. 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.
  5. 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.
  6. Lim RJ, Nik Nabil WN, Chan SY, Wong YF, Han LX, Gong JY, et al.
    Support Care Cancer, 2019 Sep;27(9):3491-3498.
    PMID: 30675667 DOI: 10.1007/s00520-019-4646-2
    PURPOSE: More than 80% head and neck cancer patients endured radiotherapy-induced xerostomia which impacts their quality of life (QoL). This observational study evaluated the effect of herbal treatment on head and neck cancer patients' xerostomia and QoL.

    METHODS: Head and neck cancer patients were recruited from July 2016 till March 2017 at National Cancer Institute, Ministry of Health, Malaysia. All study participants continued their standard oncology surveillance. Treatment group participants additionally received Chinese herbal treatment. The assessments included unstimulated salivary flow rate (USFR), stimulated salivary flow rate (SSFR), and QoL questionnaire.

    RESULTS: Of 42 recruited participants, 28 were in the treatment group and 14 were in the control group. Participants were mainly Chinese (71.4%), stage III cancer (40.5%), and had nasopharynx cancer (76.2%). The commonly used single herbs were Wu Mei, San Qi, and Tian Hua Fen. Sha Shen Mai Dong Tang, Liu Wei Di Huang Wan, and Gan Lu Yin were the frequently prescribed herbal formulas. The baseline characteristics, USFR, SSFR, and QoL between control and treatment groups were comparable (p > 0.05). USFR between control and treatment groups were similar throughout the 6-month study period. SSFR for the treatment group significantly improved from 0.15 ± 0.28 ml/min (baseline) to 0.32 ± 0.22 ml/min (p = 0.04; at the 3rd month) and subsequently achieved 0.46 ± 0.23 ml/min (p = 0.001; at the 6th month). The treatment group had better QoL in terms of speech (p = 0.005), eating (p = 0.02), and head and neck pain (p = 0.04) at the 6th month.

    CONCLUSION: Herbal treatment may improve xerostomia and QoL in post-radiotherapy head and cancer patients.

  7. Mohd-Sharif N, Shaibullah S, Givajothi V, Tan CS, Ho KL, Teh AH, et al.
    Acta Crystallogr F Struct Biol Commun, 2017 02 01;73(Pt 2):109-115.
    PMID: 28177322 DOI: 10.1107/S2053230X17001212
    TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced in Escherichia coli and purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%(v/v) ethylene glycol and 5%(v/v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space group P3121, with unit-cell parameters a = b = 126.62, c = 95.63 Å.
  8. 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.
  9. 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.

  10. 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.

  11. Ho KL, Gabrielsen M, Beh PL, Kueh CL, Thong QX, Streetley J, et al.
    PLoS Biol, 2018 Oct;16(10):e3000038.
    PMID: 30346944 DOI: 10.1371/journal.pbio.3000038
    Macrobrachium rosenbergii nodavirus (MrNV) is a pathogen of freshwater prawns that poses a threat to food security and causes significant economic losses in the aquaculture industries of many developing nations. A detailed understanding of the MrNV virion structure will inform the development of strategies to control outbreaks. The MrNV capsid has also been engineered to display heterologous antigens, and thus knowledge of its atomic resolution structure will benefit efforts to develop tools based on this platform. Here, we present an atomic-resolution model of the MrNV capsid protein (CP), calculated by cryogenic electron microscopy (cryoEM) of MrNV virus-like particles (VLPs) produced in insect cells, and three-dimensional (3D) image reconstruction at 3.3 Å resolution. CryoEM of MrNV virions purified from infected freshwater prawn post-larvae yielded a 6.6 Å resolution structure, confirming the biological relevance of the VLP structure. Our data revealed that unlike other known nodavirus structures, which have been shown to assemble capsids having trimeric spikes, MrNV assembles a T = 3 capsid with dimeric spikes. We also found a number of surprising similarities between the MrNV capsid structure and that of the Tombusviridae: 1) an extensive network of N-terminal arms (NTAs) lines the capsid interior, forming long-range interactions to lace together asymmetric units; 2) the capsid shell is stabilised by 3 pairs of Ca2+ ions in each asymmetric unit; 3) the protruding spike domain exhibits a very similar fold to that seen in the spikes of the tombusviruses. These structural similarities raise questions concerning the taxonomic classification of MrNV.
  12. Chew SY, Ho KL, Cheah YK, Ng TS, Sandai D, Brown AJP, et al.
    Sci Rep, 2019 02 26;9(1):2843.
    PMID: 30808979 DOI: 10.1038/s41598-019-39117-1
    The human fungal pathogen Candida glabrata appears to utilise unique stealth, evasion and persistence strategies in subverting the onslaught of host immune response during systemic infection. However, macrophages actively deprive the intracellular fungal pathogen of glucose, and therefore alternative carbon sources probably support the growth and survival of engulfed C. glabrata. The present study aimed to investigate the role of the glyoxylate cycle gene ICL1 in alternative carbon utilisation and its importance for the virulence of C. glabrata. The data showed that disruption of ICL1 rendered C. glabrata unable to utilise acetate, ethanol or oleic acid. In addition, C. glabrata icl1∆ cells displayed significantly reduced biofilm growth in the presence of several alternative carbon sources. It was also found that ICL1 is crucial for the survival of C. glabrata in response to macrophage engulfment. Disruption of ICL1 also conferred a severe attenuation in the virulence of C. glabrata in the mouse model of invasive candidiasis. In conclusion, a functional glyoxylate cycle is essential for C. glabrata to utilise certain alternative carbon sources in vitro and to display full virulence in vivo. This reinforces the view that antifungal drugs that target fungal Icl1 have potential for future therapeutic intervention.
  13. A Rahaman SN, Mat Yusop J, Mohamed-Hussein ZA, Aizat WM, Ho KL, Teh AH, et al.
    PeerJ, 2018;6:e5377.
    PMID: 30280012 DOI: 10.7717/peerj.5377
    Proteins of the DUF866 superfamily are exclusively found in eukaryotic cells. A member of the DUF866 superfamily, C1ORF123, is a human protein found in the open reading frame 123 of chromosome 1. The physiological role of C1ORF123 is yet to be determined. The only available protein structure of the DUF866 family shares just 26% sequence similarity and does not contain a zinc binding motif. Here, we present the crystal structure of the recombinant human C1ORF123 protein (rC1ORF123). The structure has a 2-fold internal symmetry dividing the monomeric protein into two mirrored halves that comprise of distinct electrostatic potential. The N-terminal half of rC1ORF123 includes a zinc-binding domain interacting with a zinc ion near to a potential ligand binding cavity. Functional studies of human C1ORF123 and its homologue in the fission yeast Schizosaccharomyces pombe (SpEss1) point to a role of DUF866 protein in mitochondrial oxidative phosphorylation.
  14. Pang SL, Ho KL, Waterman J, Rambo RP, Teh AH, Mathavan I, et al.
    Sci Rep, 2019 Mar 20;9(1):4933.
    PMID: 30894561 DOI: 10.1038/s41598-019-40879-x
    Group 21 and 5 allergens are homologous house dust mite proteins known as mid-tier allergens. To reveal the biological function of group 21 allergens and to understand better the allergenicity of the rDer f 21 allergen, we determined the 1.5 Å crystal structure of rDer f 21 allergen from Dermatophagoides farinae. The rDer f 21 protein consists of a three helical bundle, similar to available structures of group 21 and homologous group 5 allergens. The rDer f 21 dimer forms a hydrophobic binding pocket similar to the one in the Der p 5 allergen, which indicates that both of the homologous groups could share a similar function. By performing structure-guided mutagenesis, we mutated all 38 surface-exposed polar residues of the rDer f 21 allergen and carried out immuno-dot blot assays using 24 atopic sera. Six residues, K10, K26, K42, E43, K46, and K48, which are located in the region between the N-terminus and the loop 1 of rDer f 21 were identified as the major IgE epitopes of rDer f 21. Epitope mapping of all potential IgE epitopes on the surface of the rDer f 21 crystal structure revealed heterogeneity in the sIgE recognition of the allergen epitopes in atopic individuals. The higher the allergen-sIgE level of an individual, the higher the number of epitope residues that are found in the allergen. The results illustrate the clear correlation between the number of specific major epitope residues in an allergen and the sIgE level of the atopic population.
  15. 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.
  16. 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.
  17. 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.

  18. 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.
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