Pinyapat Kongngen’s research while affiliated with Mahidol University and other places

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Publications (1)


Schematic illustrations of recombinant EpA2 constructs. HA: HA-tag, myc myc-tag, LBD: ligand binding domain, CysRich: cysteine-rich domain, FNIII: fibronectin III repeat, Kinase: kinase domain, SAM: sterile α-motif domain, PDZ: PDZ-binding motif. The native TMD of EphA2 is depicted in blue; the PDGFR TMD is in orange. The figure was created with BioRender.com.
Transient expression of EphA2 constructs in HC-04 and the impact on P. vivax infection. (A) Western blot of the total cell lysates from HC-04 transfected with the indicated EphA2 construct. The EphA2 recombinant proteins were detected through the N-terminal HA-tag. These blots are not quantitative as different exposure were used to achieve best visual clarity. The β-actin control bands were cropped from different gels using the same respective cell lysis. (B) Representative IFA images of non-permeabilized HC-04 complemented with each EphA2 construct. EphA2 recombinant protein was detected by an HA-tag antibody (green). Nuclei (blue) were visualized with DAPI. Scale bar: 10 μm. (C) Fold increase of the liver-stage parasite infection in HC-04 expressing different EphA2 constructs. The numbers of liver-stage parasites were quantified by immunostaining with UIS4 antibody on day 4 post-infection. Error bars represent S.E.M. of 10 P. vivax clinical isolates. Different symbols represent different P. vivax clinical isolates. Asterisks mark significant differences from the mock transfection control; p-values were determined by two-way ANOVA (EphA2 construct and parasite isolate as variables) followed by Dunnett’s multiple comparisons test.
Generation of the transgenic EphA2Extra-HC04 cell line using a CRISPR-Cas9 knock-in approach. (A) Introduction of HA-Extra to the HC-04 genome by Cas9-mediated homology directed repair (HDR). Genome integration was induced by Cas9 with sgRNA targeting the AAVS1 sequence. Homologous recombination was achieved using a linear donor containing the HA-Extra cassette flanked by AAVS1 homology arms. The figure was created with BioRender.com. (B) Left: PCR demonstrating the presence of the HA-Extra sequence in two clones of the transgenic EphA2Extra-HC04 cell line. The forward primer binds to a sequence upstream of the left homology arm; the reverse primer binds to the ligand-binding domain (LBD) of the insert, resulting in a 2143-bp amplicon. Primer binding sites are depicted in (A). Right: Western blot analysis of the EphA2Extra-HC04 cell line. Total cell lysates were analyzed with the indicated antibodies. β-actin was used as the loading control. (C) Representative IFA images of EphA2Extra-HC04 clones expressing HA-Extra on the cell surface. HA-Extra was visualized with a HA-tag antibody. Nuclei were stained with DAPI. Scale bar: 10 μm.
EphA2Extra-HC04 cells enhanced P. vivax liver-stage infection. (A) Fold increase of P. vivax infection of the two EphA2Extra-HC04 clones relative to the original HC-04. Error bars represent S.E.M. of 8 biological replicates. The p-value for two-tail paired t-test between EphA2Extra-HC04 cell clones and original HC-04. (B) P. vivax-infected EphA2Extra-HC04 (top) and original HC-04 (bottom) were labeled with a HA-tag antibody (green). Liver-stage parasites were labeled with a UIS4 antibody (red). Nuclei were stained with DAPI (blue). The scale bar represents 5 μm. (C) The size distribution of liver-stage parasites in each cell line. The diameters of intrahepatic parasites on day 4 post-infection were pooled from eight P. vivax clinical isolates. Dashed lines represent the medians; dotted lines delimit the interquartile ranges. ****Indicates a p-value < 0.0001 and ns indicates a non-significant test result (p = 0.0761) by the Mann–Whitney test between the indicated pair.
Overexpression of hepatocyte EphA2 enhances liver-stage infection by Plasmodium vivax
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December 2022

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1 Citation

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Ubonwan Jaihan

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The liver is the first destination of malaria parasites in humans. After reaching the liver by the blood stream, Plasmodium sporozoites cross the liver sinusoid epithelium, enter and exit several hepatocytes, and eventually invade a final hepatocyte host cell. At present, the mechanism of hepatocyte invasion is only partially understood, presenting a key research gap with opportunities for the development of new therapeutics. Recently, human EphA2, a membrane-bound receptor tyrosine kinase, was implicated in hepatocyte infection by the human malaria parasite Plasmodium falciparum and the rodent parasite Plasmodium yoelii, but its role is not known for Plasmodium vivax, a major human parasite whose liver infection poses a specific challenge for malaria treatment and elimination. In this study, the role of EphA2 in P. vivax infection was investigated. It was found that surface expression of several recombinant fragments of EphA2 enhanced the parasite infection rate, thus establishing its role in P. vivax infection. Furthermore, a new permanent cell line (EphA2Extra-HC04) expressing the whole extracellular domain of EphA2 was generated. This cell line supports a higher rate of P. vivax infection and is a valuable tool for P. vivax liver-stage research.

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Citations (1)


... Remarkably, we noted that ALW-II-41-27 could indeed significantly reduce TNF-alpha protein levels in the lungs versus the vehicle control group in yeast β-glucan-challenged mouse lungs (Fig. 6). A number of reports have shown the importance of the EphA2 receptor pathway in organism attachment and host immune recognition to microbial pathogens (12,(33)(34)(35). Recently, we also have reported that EphA2 can bind Pneumocystis glucans and is involved in lung epithelial cell proinflammatory response to the organism's cell wall carbohydrate (1). ...

Reference:

Targeting host tyrosine kinase receptor EphA2 signaling via small-molecule ALW-II-41-27 inhibits macrophage pro-inflammatory signaling responses to Pneumocystis carinii β-glucans
Overexpression of hepatocyte EphA2 enhances liver-stage infection by Plasmodium vivax