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Fulltext Response of Cellular Innate Immunity to Cnidarian Pore-Forming Toxins

Yap WY, Hwang JS

Molecules, 2018 Oct 04;23(10).
PMID: 30287801 DOI: 10.3390/molecules23102537

A group of stable, water-soluble and membrane-bound proteins constitute the pore forming toxins (PFTs) in cnidarians. They interact with membranes to physically alter the membrane structure and permeability, resulting in the formation of pores. These lesions on the plasma membrane causes an imbalance of cellular ionic gradients, resulting in swelling of the cell and eventually its rupture. Of all cnidarian PFTs, actinoporins are by far the best studied subgroup with established knowledge of their molecular structure and their mode of pore-forming action. However, the current view of necrotic action by actinoporins may not be the only mechanism that induces cell death since there is increasing evidence showing that pore-forming toxins can induce either necrosis or apoptosis in a cell-type, receptor and dose-dependent manner. In this review, we focus on the response of the cellular immune system to the cnidarian pore-forming toxins and the signaling pathways that might be involved in these cellular responses. Since PFTs represent potential candidates for targeted toxin therapy for the treatment of numerous cancers, we also address the challenge to overcoming the immunogenicity of these toxins when used as therapeutics.
Fulltext Is Proteolytic Cleavage Essential for the Enhanced Activity of Hydra Pore-Forming Toxin, HALT-4?

Yap WY, Hwang JS

Toxins (Basel), 2023 Jun 13;15(6).
PMID: 37368697 DOI: 10.3390/toxins15060396

Hydra actinoporin-like toxin 4 (HALT-4) differs from other actinoporins due to its N-terminal propart that contains approximately 103 additional residues. Within this region, we identified five dibasic residues and assumed that, when cleaved, they could potentially exhibit HALT-4's cytolytic activity. We created five truncated versions of HALT-4 (tKK1, tKK2, tRK3, tKK4 and tKK5) to investigate the role of the N-terminal region and potential cleavage sites on the cytolytic activity of HALT-4. However, our results demonstrated that the propart-containing HALT-4 (proHALT-4), as well as the truncated versions tKK1 and tKK2, exhibited similar cytolytic activity against HeLa cells. In contrast, tRK3, tKK4 and tKK5 failed to kill HeLa cells, indicating that cleavage at the KK1 or KK2 sites did not enhance cytolytic activity but may instead facilitate the sorting of tKK1 and tKK2 to the regulated secretory pathway for eventual deposition in nematocysts. Moreover, RK3, KK4 and KK5 were unlikely to serve as proteolytic cleavage sites, as the amino acids between KK2 and RK3 are also crucial for pore formation.
Expansion of Hydra actinoporin-like toxin (HALT) gene family: Expression divergence and functional convergence evolved through gene duplication

Yap WY, Tan KJSX, Hwang JS

Toxicon, 2019 Dec;170:10-20.
PMID: 31513812 DOI: 10.1016/j.toxicon.2019.09.007

Hydra actinoporin-like toxin 1 (HALT-1) was previously shown to cause cytolysis and haemolysis in a number of human cells and has similar functional properties to the actinoporins equinatoxin and sticholysin. In addition to HALT-1, five other HALTs (HALTs 2, 3, 4, 6 and 7) were also isolated from Hydra magnipapillata and expressed as recombinant proteins in this study. We demonstrated that recombinant HALTs have cytolytic activity on HeLa cells but each exhibited a different range of toxicity. All six recombinant HALTs bound to sulfatide, while rHALT-1 and rHALT-3 bound to two additional sphingolipids, lysophosphatidic acid and sphingosine-1-phosphate as indicated by the protein-lipid overlay assay. When either tryptophan133 or tyrosine129 of HALT-1 was mutated, the mutant protein lost binding to sulfatide, lysophosphatidic acid and sphingosine-1-phosphate. As further verification of HALTs' binding to sulfatide, we performed ELISA for each HALT. To determine the cell-type specific gene expression of seven HALTs in Hydra, we searched for individual HALT expression in the single-cell RNA-seq data set of Single Cell Portal. The results showed that HALT-1, 4 and 7 were expressed in differentiating stenoteles. HALT-1 and HALT-6 were expressed in the female germline during oogenesis. HALT-2 was strongly expressed in the gland and mucous cells in the endoderm. Information on HALT-3 and HALT-5 could not be found in the single-cell data set. Our findings show that subfunctionalisation of gene expression following duplication enabled HALTs to become specialized in various cell types of the interstitial cell lineage.
Not all swellings are lymph nodes! A case of subcutaneous panniculitis-like T-cell lymphoma

Tee Evelyn Wy Yap CT, Evelyn Yap WY

Med J Malaysia, 2019 Oct;74(5):441-442.
PMID: 31649225

Subcutaneous Panniculitis-like T-cell Lymphoma (SPTL) is a rare cutaneous neoplasm of mature cytotoxic T cells, first described in 1991 by Gonzalez et al. The incidence of SPTL in Asian countries ranges from 2.3% to 3%. In Malaysia, only 5 cases were reported from 2001 to 2004 in Hospital Kuala Lumpur, Malaysia. SPTL typically presents as skincoloured or erythematous subcutaneous nodules, most often on the extremities and trunk, but it can also involve the face, back and neck. Diagnosis of SPTL is made based on correlation of clinical findings and subcutaneous tissue biopsy along with immunohistochemical staining patterns.
Enhanced production of recombinant HALT-1 pore-forming toxin using two-step chromatographic procedure

Yap WY, Loo LW, Sha HX, Hwang JS

MethodsX, 2023;10:102073.
PMID: 36865650 DOI: 10.1016/j.mex.2023.102073

Hydra actinoporin-like toxin-1 (HALT-1) has been isolated from Hydra magnipapillata and is highly cytolytic against various human cells including erythrocyte. Previously, recombinant HALT-1 (rHALT-1) was expressed in Escherichia coli and purified by the nickel affinity chromatography. In this study, we improved the purification of rHALT-1 by two-step purifications. Bacterial cell lysate containing rHALT-1 was subjected to the sulphopropyl (SP) cation exchange chromatography with different buffers, pHs, and NaCl concentrations. The results indicated that both phosphate and acetate buffers facilitated the strong binding of rHALT-1 to SP resins, and the buffers containing 150 mM and 200 mM NaCl, respectively, removed protein impurities but retain most rHALT-1 in the column. When combining the nickel affinity chromatography and the SP cation exchange chromatography, the purity of rHALT-1 was highly enhanced. In subsequent cytotoxicity assays, 50% of cells could be lysed at ∼18 and ∼22 µg/mL of rHALT-1 purified with phosphate and acetate buffers, respectively.•HALT-1 is a soluble α-pore-forming toxin of 18.38 kDa.•rHALT-1 was purified by nickel affinity chromatography followed by SP cation exchange chromatography.•The cytotoxicity of purified rHALT-1 using 2-step purifications via either phosphate or acetate buffer was comparable to those previously reported.
Fulltext An in vitro Comparison of Bond Strength of Different Sealers/Obturation Systems to Root Dentin Using the Push-Out Test at 2 Weeks and 3 Months after Obturation

Yap WY, Che Ab Aziz ZA, Azami NH, Al-Haddad AY, Khan AA

Med Princ Pract, 2017;26(5):464-469.
PMID: 28934753 DOI: 10.1159/000481623

OBJECTIVE: To evaluate the push-out bond strength and failure modes of different sealers/obturation systems to intraradicular dentin at 2 weeks and 3 months after obturation compared to AH Plus®/gutta-percha.
MATERIALS AND METHODS: A total of 180 root slices from 60 single-canal anterior teeth were prepared and assigned to 5 experimental groups (n = 36 in each group), designated as G1 (AH Plus®/gutta-percha), G2 (TotalFill BC™ sealer/BC-coated gutta-percha), G3 (TotalFill BC™ sealer/gutta-percha), G4 (EndoREZ® sealer/EndoREZ®-coated gutta-percha), and G5 (EndoREZ® sealer/gutta-percha). Push-out bond strengths of 18 root slices in each group were assessed at 2 weeks and the other 18 at 3 months after obturation using a universal testing machine. Data were analyzed using repeated measures ANOVA. An independent t test was used to compare the mean push-out bond strength for each group at 2 weeks and 3 months after obturation.
RESULTS: The mean push-out bond strengths of G4 and G5 were significantly lower than those of G1, G2, and G3 (p < 0.05) at both 2 weeks (G1: 1.46 ± 0.29 MPa, G2: 1.74 ± 0.43 MPa, G3: 1.74 ± 0.43 MPa, G4: 0.66 ± 0.31 MPa, G5: 0.74 ± 0.47 MPa) and 3 months after obturation (G1: 1.70 ± 1.05 MPa, G2: 3.69 ± 1.20 MPa, G3: 2.84 ± 0.83 MPa, G4: 0.14 ± 0.05 MPa, G5: 0.24 ± 0.10 MPa). The mean push-out bond strengths of G2 (3.69 ± 1.20 MPa) and G3 (2.84 ± 0.83 MPa) were higher at 3 months compared to 2 weeks after obturation (G2: 1.74 ± 0.43 MPa, G3: 1.33 ± 0.29 MPa).
CONCLUSION: The TotalFill BC™ obturation system (G2) and the TotalFill BC™ sealer/gutta-percha (G3) showed comparable bond strength to AH Plus®. Their bond strength increased over time, whereas the EndoREZ® obturation system (G4) and EndoREZ sealer (G5) had low push-out bond strength which decreased over time.