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Preclinical assessment of VPEAV, a new trivalent antivenom for elapid snakebite envenoming in the Philippines: Proteomics, immunoreactivity and toxicity neutralization

Chan YW, Tan KY, Tan CH

Toxicon, 2022 Dec;220:106942.
PMID: 36240856 DOI: 10.1016/j.toxicon.2022.106942

Snakebite envenoming is an important neglected tropical disease. Antivenom supply, however, remains limited in many parts of the world. This study aimed to examine the protein composition, immunoreactivity and neutralization efficacy of a new antivenom product (VINS Philippine Elapid Antivenoms, VPEAV) developed for the treatment of snakebite envenoming caused by the Philippine Cobra (Naja philippinensis), Samar Cobra (Naja samarensis) and King Cobra (Ophiophagus hannah). Size-exclusion chromatography, sodium-dodecyl sulfate-polyacrylamide gel electrophoresis and tandem mass spectrometry showed that VPEAV consisted of F(ab)'2 (∼90% of total antivenom proteins) with minimal protein impurities. Indirect ELISA showed varying immunoreactivity of VPEAV toward the different venoms (EC50 = 4-16 μg/ml), indicating distinct venom antigenicity between the species. In mice, the neutralization potency of VPEAV against the King Cobra venom was moderate (potency, P = 2.6 mg/ml, defined as the amount of venom completely neutralized per unit volume of antivenom). The potency was significantly lower against the N. philippinensis and N. samarensis venoms (P = 0.18-0.30 mg/ml), implying a higher dose may be needed for effective neutralization of the Naja venoms. Together, the findings suggest the potential and limitation of VPEAV in neutralizing the venom toxicity of the three Philippine elapid snakes.
Structure-informed separation of bioactive peptides

Acquah C, Chan YW, Pan S, Agyei D, Udenigwe CC

J Food Biochem, 2019 01;43(1):e12765.
PMID: 31353493 DOI: 10.1111/jfbc.12765

The application of proteomic and peptidomic technologies for food-derived bioactive peptides is an emerging field in food sciences. These technologies include the use of separation tools coupled to a high-resolution spectrometric and bioinformatic tools for prediction, identification, sequencing, and characterization of peptides. To a large extent, one-dimensional separation technologies have been extensively used as a continuous tool under different optimized conditions for the identification and analysis of food peptides. However, most one-dimensional separation technologies are fraught with significant bottlenecks such as insufficient sensitivity and specificity limits for complex samples. To address this limitation, separation systems based on orthogonal, multidimensional principles, which allow for the coupling of more than one analytical separation tool with different operational principles, provide a higher separation power than one-dimensional separation tools. This review describes the structure-informed separation and purification of protein hydrolyzates to obtain peptides with desirable bioactivities. PRACTICAL APPLICATIONS: Application of bioactive peptides in the formulation of functional foods, nutraceuticals, and therapeutic agents have increasingly gained scholarly and industrial attention. The bioactive peptides exist originally in protein sources and are only active after hydrolysis of the parent protein. Currently, several tools can be configured in one-dimensional or multidimensional systems for the separation and purification of protein hydrolyzates. The separations are informed by the structural properties such as the molecular weight, charge, hydrophobicity or hydrophilicity, and the solubility of peptides. This review provides a concise discussion on the commonly used analytical tools, their configurations, advantages and challenges in peptide separation. Emphasis is placed on how the structural properties of peptides assist in the separation and purification processes and the concomitant effect of the separation on peptide bioactivity.
Fulltext Surgical reduction technique (transpedicle) for unstable thoracolumbar burst fractures with retropulsion resulting in severe spinal canal stenosis: a preliminary case report

Choo CH, Kwan MK, Chris Chan YW

AME Case Rep, 2018;2:38.
PMID: 30264034 DOI: 10.21037/acr.2018.07.02

Thoracolumbar burst fractures are common entity in polytraumatized patients. The retropulsed burst vertebral fracture may result in spinal canal invasion with or without neurological deficit. In this situation, early surgical stabilization with decompression is vital to restore neurological function. We employed a posterior approach with a unique transpedicular reduction technique at the level of fracture for decompression and stabilisation.
Fulltext Snake Venom Proteomics of Samar Cobra (Naja samarensis) from the Southern Philippines: Short Alpha-Neurotoxins as the Dominant Lethal Component Weakly Cross-Neutralized by the Philippine Cobra Antivenom

Palasuberniam P, Chan YW, Tan KY, Tan CH

Front Pharmacol, 2021;12:727756.
PMID: 35002690 DOI: 10.3389/fphar.2021.727756

The Samar Cobra, Naja samarensis, is endemic to the southern Philippines and is a WHO-listed Category 1 venomous snake species of medical importance. Envenomation caused by N. samarensis results in neurotoxicity, while there is no species-specific antivenom available for its treatment. The composition and neutralization of N. samarensis venom remain largely unknown to date. This study thus aimed to investigate the venom proteome of N. samarensis for a comprehensive profiling of the venom composition, and to examine the immunorecognition as well as neutralization of its toxins by a hetero-specific antivenom. Applying C18 reverse-phase high-performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (LC-MS/MS), three-finger toxins (3FTx) were shown to dominate the venom proteome by 90.48% of total venom proteins. Other proteins in the venom comprised snake venom metalloproteinases, phospholipases A2, cysteine-rich secretory proteins, venom nerve growth factors, L-amino acid oxidases and vespryn, which were present at much lower abundances. Among all, short-chain alpha-neurotoxins (SαNTX) were the most highly expressed toxin within 3FTx family, constituting 65.87% of the total venom proteins. The SαNTX is the sole neurotoxic component of the venom and has an intravenous median lethal dose (LD50) of 0.18 μg/g in mice. The high abundance and low LD50 support the potent lethal activity of N. samarensis venom. The hetero-specific antivenom, Philippine Cobra Antivenom (PCAV, raised against Naja philippinensis) were immunoreactive toward the venom and its protein fractions, including the principal SαNTX. In efficacy study, PCAV was able to cross-neutralize the lethality of SαNTX albeit the effect was weak with a low potency of 0.20 mg/ml (defined as the amount of toxin completely neutralized per milliliter of the antivenom). With a volume of 5 ml, each vial of PCAV may cross-neutralize approximately 1 mg of the toxin in vivo. The findings support the potential para-specific use of PCAV in treating envenomation caused by N. samarensis while underscoring the need to improve the potency of its neutralization activity, especially against the highly lethal alpha-neurotoxins.
Fulltext Characterisation of aptamer-anchored poly(EDMA-co-GMA) monolith for high throughput affinity binding

Acquah C, Chan YW, Pan S, Yon LS, Ongkudon CM, Guo H, et al.

Sci Rep, 2019 10 10;9(1):14501.
PMID: 31601836 DOI: 10.1038/s41598-019-50862-1

Immobilisation of aptameric ligands on solid stationary supports for effective binding of target molecules requires understanding of the relationship between aptamer-polymer interactions and the conditions governing the mass transfer of the binding process. Herein, key process parameters affecting the molecular anchoring of a thrombin-binding aptamer (TBA) onto polymethacrylate monolith pore surface, and the binding characteristics of the resulting macroporous aptasensor were investigated. Molecular dynamics (MD) simulations of the TBA-thrombin binding indicated enhanced Guanine 4 (G4) structural stability of TBA upon interaction with thrombin in an ionic environment. Fourier-transform infrared spectroscopy and thermogravimetric analyses were used to characterise the available functional groups and thermo-molecular stability of the immobilised polymer generated with Schiff-base activation and immobilisation scheme. The initial degradation temperature of the polymethacrylate stationary support increased with each step of the Schiff-base process: poly(Ethylene glycol Dimethacrylate-co-Glycidyl methacrylate) or poly(EDMA-co-GMA) [196.0 °C (±1.8)]; poly(EDMA-co-GMA)-Ethylenediamine [235.9 °C (±6.1)]; poly(EDMA-co-GMA)-Ethylenediamine-Glutaraldehyde [255.4 °C (±2.7)]; and aptamer-modified monolith [273.7 °C (±2.5)]. These initial temperature increments reflected in the associated endothermic energies were determined with differential scanning calorimetry. The aptameric ligand density obtained after immobilisation was 480 pmol/μL. Increase in pH and ionic concentration affected the surface charge distribution and the binding characteristics of the aptamer-modified disk-monoliths, resulting in the optimum binding pH and ionic concentration of 8.0 and 5 mM Mg2+, respectively. These results are critical in understanding and setting parametric constraints indispensable to develop and enhance the performance of aptasensors.
Plasma polymerized carvone as an antibacterial and biocompatible coating

Chan YW, Siow KS, Ng PY, Gires U, Yeop Majlis B

Mater Sci Eng C Mater Biol Appl, 2016 Nov 01;68:861-871.
PMID: 27524089 DOI: 10.1016/j.msec.2016.07.040

Antibacterial coating is important to prevent the colonization of medical devices by biofilm forming bacteria that would cause infection and sepsis in patients. Current coating techniques such as immobilization of antimicrobial compounds, time-releasing antibiotic agents and silver nanoparticles, require multiple processing steps, and they have low efficacy and low stability. We proposed a single-step plasma polymerization of an essential oil known as carvone to produce a moderately hydrophobic antibacterial coating (ppCar) with an average roughness of <1nm. ppCar had a static water contact angle of 78°, even after 10days of air aging and it maintained its stability throughout 24h of LB broth immersion. ppCar showed promising results in the live-dead fluorescence assay and crystal violet assay. The biofilm assay showed an effective reduction of E. coli and S. aureus bacteria by 86% and 84% respectively. ppCar is also shown to rupture the bacteria membrane for its bactericidal effects. The cytotoxicity test indicated that the coating is not cytotoxic to the human cell line. This study would be of interest to researcher keen on producing a bacteria-resistance and biocompatible coating on different substrates in a cost-effective manner.
Parametric study of immobilized cellulase-polymethacrylate particle for the hydrolysis of carboxymethyl cellulose

Chan YW, Acquah C, Obeng EM, Dullah EC, Jeevanandam J, Ongkudon CM

Biochimie, 2019 Feb;157:204-212.
PMID: 30513369 DOI: 10.1016/j.biochi.2018.11.019

Biocarriers are pivotal in enhancing the reusability of biocatalyst that would otherwise be less economical for industrial application. Ever since the induction of enzymatic technology, varied materials have been assessed for their biocompatibility with enzymes of distinct functionalities. Herein, cellulase was immobilized onto polymethacrylate particles (ICP) as the biocarrier grafted with ethylenediamine (EDA) and glutaraldehyde (GA). Carboxymethyl cellulose (CMC) was used as a model substrate for activity assay. Enzyme immobilization loading was determined by quantifying the dry weight differential of ICP (pre-& post-immobilization). Cellulase was successfully demonstrated to be anchored upon ICP and validated by FTIR spectra analysis. The optimal condition for cellulase immobilization was determined to be pH 6 at 20 °C. The maximum CMCase activity was achieved at pH 5 and 50 °C. Residual activity of ∼50% was retained after three iterations and dipped to ∼18% on following cycle. Also, ICP displayed superior pH adaptability as compared to free cellulase. The specific activity of ICP was 65.14 ± 1.11% relative to similar amount of free cellulase.
A 5-year retrospective study of melioidosis cases treated in a district specialist hospital

Tang RY, Lim SH, Lam JE, Nurasykin S, Eileen T, Chan YW

Med J Malaysia, 2019 12;74(6):472-476.
PMID: 31929471

INTRODUCTION: Melioidosis is caused by Burkholderia pseudomallei, a gram-negative aerobic bacillus, found in the soil and surface water. Treating melioidosis has been a challenge in district hospitals due to high usage of broad spectrum antibiotics and prolonged hospitalisation. This study is to review the patients' demography, clinical presentations and microbiological data.
METHODS: A 5-year retrospective study was carried out on patients admitted with culture positive for melioidosis from year 2013 to 2017 in Hospital Teluk Intan, Perak.
RESULTS: There were a total of 46 confirmed cases of melioidosis. Majority of the patients were working in the agricultural and farming (28.6%), and factories (25.7%). Thirty-one patients had diabetes mellitus (71.1%). Presentations of patients with melioidosis included pneumonia (54.3%), skin and soft tissue infection (19.6%), deep abscesses (15.2%) and bone and joint infections (13%). An average of 5.8 days was needed to confirm the diagnosis of melioidosis via positive culture. However, only 39.4% of these patients were started on ceftazidime or carbapenem as the empirical therapy. The intensive care unit (ICU) admission rate for melioidosis was 46% and the mortality rate was 52%. Our microbial cultures showed good sensitivity towards cotrimoxazole (97.1%), ceftazidime (100%) and carbapenem (100%).
CONCLUSION: Melioidosis carries high mortality rate, especially with lung involvement and bacteremia. Physicians should have high clinical suspicion for melioidosis cases to give appropriate antimelioidosis therapy early.
Decomplexation proteomic analysis and purity assessment of a biologic for snakebite envenoming: Philippine Cobra Antivenom

Palasuberniam P, Tan KY, Chan YW, Blanco FB, Tan CH

Trans R Soc Trop Med Hyg, 2023 Jun 02;117(6):428-434.
PMID: 36611268 DOI: 10.1093/trstmh/trac125

BACKGROUND: Philippine Cobra Antivenom (PCAV) is the only snake antivenom manufactured in the Philippines. It is used clinically to treat envenoming caused by the Philippine Spitting Cobra (Naja philippinensis). While PCAV is effective pharmacologically, it is crucial to ensure the safety profile of this biologic that is derived from animal plasma.
METHODS: This study examined the composition purity of PCAV through a decomplexation proteomic approach, applying size-exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tandem mass spectrometry liquid chromatography-tandem mass spectrometry (LC-MS/MS).
RESULTS: SDS-PAGE and SEC showed that the major protein in PCAV (constituting ∼80% of total proteins) is approximately 110 kDa, consistent with the F(ab')2 molecule. This protein is reducible into two subunits suggestive of the light and heavy chains of immunoglobulin G. LC-MS/MS further identified the proteins as equine immunoglobulins, representing the key therapeutic ingredient of this biologic product. However, protein impurities, including fibrinogens, alpha-2-macroglobulins, albumin, transferrin, fibronectin and plasminogen, were detected at ∼20% of the total antivenom proteins, unveiling a concern for hypersensitivity reactions.
CONCLUSIONS: Together, the findings show that PCAV contains a favorable content of F(ab')2 for neutralization, while the antibody purification process awaits improvement to minimize the presence of protein impurities.