a-competent-hepatocyte-model-examining-hepatitis-b-virus-entry

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

Mahidol University

A rhodol cinnamate fluorescent chemosensor (RC) has been developed for selective detection of hydrazine (NH). In aqueous medium, the rhodol-based probe exhibited high selectivity for hydrazine among other molecules. The addition of hydrazine triggered a fluorescence emission with 48-fold enhancement based on hydrazinolysis and a subsequent ring-opening process. The chemical probe also displayed a selective colorimetric response toward NH from colorless solution to pink, readily observed by the naked eye. The detection limit of RC for hydrazine was calculated to be 300nM (9.6ppb). RC is membrane permeable and was successfully demonstrated to detect hydrazine in live HepG2 cells by confocal fluorescence microscopy.

A rhodol-based fluorescent chemosensor for hydrazine and its application in live cell bioimaging.

A rhodamine-based fluorescent chemodosimeter rhodamine hydrazide-triazole (RHT) tethered with a triazole moiety was developed for Cu detection. In aqueous medium, the RHT probe exhibited high selectivity and sensitivity toward Cu among other metal ions. The addition of Cu triggered a fluorescence emission of RHT by 384-fold (Φ = 0.33) based on a ring-opening process and a subsequent hydrolysis reaction. Moreover, RHT also showed a selective colorimetric response toward Cu from colorless solution to pink, readily observed with the naked eye. The limit of detection of RHT for Cu was calculated to be 1 nM (0.06 ppb). RHT was successfully demonstrated to detect Cu in Chang liver cells by confocal fluorescence microscopy.

A rhodamine-triazole fluorescent chemodosimeter for Cu detection and its application in bioimaging.

Eradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax, to cause relapse in patients. Research efforts to better understand the biology of P. vivax hypnozoite and design relapse prevention strategies have been hampered by the lack of a robust and reliable model for in vitro culture of liver-stage parasites. Although the HC-04 hepatoma cell line is used for culturing liver-stage forms of Plasmodium, these cells proliferate unrestrictedly and detach from the culture dish after several days, which limits their usefulness in a long-term hypnozoite assay.

A novel immortalized hepatocyte-like cell line (imHC) supports in vitro liver stage development of the human malarial parasite Plasmodium vivax.

A rhodol-based fluorescent probe has been developed as a selective hydrazine chemosensor using levulinate as a recognition site. The rhodol levulinate probe (RL) demonstrated high selectivity and sensitivity toward hydrazine among other molecules. The chromogenic response of RL solution to hydrazine from colorless to pink could be readily observed by the naked eye, while strong fluorescence emission could be monitored upon excitation at 525 nm. The detection process occurred via a ring-opening process of the spirolactone initiated by hydrazinolysis, triggering the fluorescence emission with a 53-fold enhancement. The probe rapidly reacted with hydrazine in aqueous medium with the detection limit of 26 nM (0.83 ppb), lower than the threshold limit value (TLV) of 10 ppb suggested by the U.S. Environmental Protection Agency. Furthermore, RL-impregnated paper strips could detect hydrazine vapor. For biological applicability of RL, its membrane-permeable property led to bioimaging of hydrazine in live HepG2 cells by confocal fluorescence microscopy.

A chromogenic and fluorogenic rhodol-based chemosensor for hydrazine detection and its application in live cell bioimaging.

More than 350 million people worldwide have been persistently infected with the hepatitis B virus (HBV). Chronic HBV infection could advance toward liver cirrhosis and hepatocellular carcinoma. The intervention with prophylactic vaccine and conventional treatment could suppress HBV, but could not completely eradicate it. The major obstacle for investigating curative antiviral drugs are the incompetence of hepatocyte models that should have closely imitated natural human infection. Here, we demonstrated that an immortalized hepatocyte-like cell line (imHC) could accommodate for over 30 days the entire life cycle of HBV prepared from either established cultured cells or clinically-derived fresh isolates. Normally, imHCs had intact interferon signaling with anti-viral action. Infected imHCs responded to treatments with direct-acting antiviral drugs (DAAs) and interferons (IFNs) by diminishing HBV DNA, the covalently closed circular DNA (cccDNA) surface antigen of HBV (HBsAg, aka the Australia antigen) and the hepatitis B viral protein (HBeAg). Notably, we could observe and quantify HBV spreading from infected cells to naïve cells using an imHC co-culture model. In summary, this study constructed a convenient HBV culture model that allows the screening for novel anti-HBV agents with versatile targets, either HBV entry, replication or cccDNA formation. Combinations of agents aiming at different targets should achieve a complete HBV eradication.

An Immortalized Hepatocyte-like Cell Line (imHC) Accommodated Complete Viral Lifecycle, Viral Persistence Form, cccDNA and Eventual Spreading of a Clinically-Isolated HBV.

Double-stranded RNA (dsRNA) is employed to down-regulate the expression of specific genes of shrimp viral pathogens through the RNA interference (RNAi) pathway. The administration of dsRNA into shrimp has been shown to be an effective strategy to block yellow head virus (YHV) progression. In this study, a vector (pLVX-AcGFP1-N1) was developed to introduce a long-hairpin RNA (lhRNA) silencing cassette under a CMV promoter, so-called "pLVX-lhRdRp", against the RNA-dependent RNA polymerase (RdRp) gene of YHV. A primary culture of hemocytes isolated from Penaeus monodon was transfected with the pLVX-lhRdRp vector, generating transcripts of lhRNAs as early as 12 h post transfection. Twelve hours prior to YHV challenge, the primary hemocyte cell culture was transfected with pLVX-lhRdRp, whereas control groups were transfected with pLVX-AcGFP1-N1 or no transfection. The group treated with pLVX-lhRdRp significantly suppressed YHV replication at 24-72 h after YHV challenge. The results from RT-PCR and immunohistochemistry confirmed that both mRNA and protein expression of YHV were effectively inhibited by the pLVX-lhRdRp vector. Thus, our hemocyte culture and dsRNA expression plasmid with constitutive promoter have potential as a platform to test DNA constructs expressing long-hairpin RNA against pathogenic viral infection and as a RNAi-based DNA vaccine in shrimp.

Effective suppression of yellow head virus replication in Penaeus monodon hemocytes using constitutive expression vector for long-hairpin RNA (lhRNA).

Since December 2019, the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused severe pneumonia, a disease named COVID-19, that became pandemic and created an acute threat to public health. The effective therapeutics are in urgent need. Here, we developed a high-content screening for the antiviral candidates using fluorescence-based SARS-CoV-2 nucleoprotein detection in Vero E6 cells coupled with plaque reduction assay. Among 122 Thai natural products, we found that Boesenbergia rotunda extract and its phytochemical compound, panduratin A, exhibited the potent anti-SARS-CoV-2 activity. Treatment with B. rotunda extract and panduratin A after viral infection drastically suppressed SARS-CoV-2 infectivity in Vero E6 cells with IC of 3.62 μg/mL (CC = 28.06 µg/mL) and 0.81 μΜ (CC = 14.71 µM), respectively. Also, the treatment of panduratin A at the pre-entry phase inhibited SARS-CoV-2 infection with IC of 5.30 µM (CC = 43.47 µM). Our study demonstrated, for the first time, that panduratin A exerts the inhibitory effect against SARS-CoV-2 infection at both pre-entry and post-infection phases. Apart from Vero E6 cells, treatment with this compound was able to suppress viral infectivity in human airway epithelial cells. This result confirmed the potential of panduratin A as the anti-SARS-CoV-2 agent in the major target cells in human. Since B. rotunda is a culinary herb generally grown in China and Southeast Asia, its extract and the purified panduratin A may serve as the promising candidates for therapeutic purposes with economic advantage during COVID-19 situation.

High-content screening of Thai medicinal plants reveals Boesenbergia rotunda extract and its component Panduratin A as anti-SARS-CoV-2 agents.

Suitable cell models are essential to advance our understanding of the pathogenesis of liver diseases and the development of therapeutic strategies. Primary human hepatocytes (PHHs), the most ideal hepatic model, are commercially available, but they are expensive and vary from lot-to-lot which confounds their utility. We have recently developed an immortalized hepatocyte-like cell line (imHC) from human mesenchymal stem cells, and tested it for use as a substitute model for hepatotropic infectious diseases. With a special interest in liver pathogenesis of viral infection, herein we determined the suitability of imHC as a host cell target for dengue virus (DENV) and as a model for anti-viral drug testing. We characterized the kinetics of DENV production, cellular responses to DENV infection (apoptosis, cytokine production and lipid droplet metabolism), and examined anti-viral drug effects in imHC cells with comparisons to the commonly used hepatoma cell lines (HepG2 and Huh-7) and PHHs. Our results showed that imHC cells had higher efficiencies in DENV replication and NS1 secretion as compared to HepG2 and Huh-7 cells. The kinetics of DENV infection in imHC cells showed a slower rate of apoptosis than the hepatoma cell lines and a certain similarity of cytokine profiles to PHHs. In imHC, DENV-induced alterations in levels of lipid droplets and triacylglycerols, a major component of lipid droplets, were more apparent than in hepatoma cell lines, suggesting active lipid metabolism in imHC. Significantly, responses to drugs with DENV inhibitory effects were greater in imHC cells than in HepG2 and Huh-7 cells. In conclusion, our findings suggest superior suitability of imHC as a new hepatocyte model for studying mechanisms underlying viral pathogenesis, liver diseases and drug effects.

Immortalized stem cell-derived hepatocyte-like cells: An alternative model for studying dengue pathogenesis and therapy.

The coronaviruses disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has become a major health problem, affecting more than 50 million people with over one million deaths globally. Effective antivirals are still lacking. Here, we optimized a high-content imaging platform and the plaque assay for viral output study using the legitimate model of human lung epithelial cells, Calu-3, to determine the anti-SARS-CoV-2 activity of  extract and its major component, andrographolide. SARS-CoV-2 at 25TCID was able to reach the maximal infectivity of 95% in Calu-3 cells. Postinfection treatment of  and andrographolide in SARS-CoV-2-infected Calu-3 cells significantly inhibited the production of infectious virions with an IC of 0.036 μg/mL and 0.034 μM, respectively, as determined by the plaque assay. The cytotoxicity profile developed over the cell line representatives of major organs, including liver (HepG2 and imHC), kidney (HK-2), intestine (Caco-2), lung (Calu-3), and brain (SH-SY5Y), showed a CC of >100 μg/mL for  extract and 13.2-81.5 μM for andrographolide, respectively, corresponding to a selectivity index of over 380. In conclusion, this study provided experimental evidence in favor of  and andrographolide for further development as a monotherapy or in combination with other effective drugs against SARS-CoV-2 infection.

Anti-SARS-CoV-2 Activity of  Extract and Its Major Component Andrographolide in Human Lung Epithelial Cells and Cytotoxicity Evaluation in Major Organ Cell Representatives.

A highly selective rhodamine hydrazide-based fluorescent chemosensor for Au detection was developed. The aqueous solution of rhodamine N-hydroxysemicarbazide (RHS), in the presence of Au, exhibited a significant 55-fold turn-on fluorescence response at 591 nm and a colorimetric change from colorless to pink. Other interested ions including Li, Na, K, Cs, Mg, Ca, Ba, Pb, Mn, Co, Ni, Ag, Cd, Cu, Hg, Zn, Sn, Fe, Fe, Cr, Ce did not induce any distinct color/spectral changes. The irreversible detection mechanism occurred via Au-promoted 5-exo-trig ring closure to yield 1,3,4-oxadiazole-2-one product. The RHS probe is non-responsive to other biologically relevant metal ions and the limit of detection for Au was calculated to be 0.5 µM with a linear range of 0 to 90 µM. Fluorescence bioimaging of Au in HepG2 cells was also successfully demonstrated.

A Rhodamine-based Fluorescent Chemodosimeter for Au in Aqueous Solution and Living Cells.

Hepatitis B virus (HBV) has been implicated in hepatitis and hepatocellular carcinoma. Current agents (nucleos(t)ide analogs and interferons) could only attenuate HBV infection. A combination of agents targeting different stages of viral life cycle (e.g., entry, replication, and cccDNA stability) was expected to eradicate the infection. Curcumin (CCM) was investigated for inhibitory action toward HBV attachment and internalization. Immortalized hepatocyte-like cells (imHCs), HepaRG and non-hepatic cells served as host cells for binding study with CCM. CCM decreased viral load, HBeAg, HBcAg (infectivity), intracellular HBV DNA, and cccDNA levels. The CCM-induced suppression of HBV entry was directly correlated with the density of sodium-taurocholate co-transporting polypeptide (NTCP), a known host receptor for HBV entry. The site of action of CCM was confirmed using TCA uptake assay. The affinity between CCM and NTCP was measured using isothermal titration calorimetry (ITC). These results demonstrated that CCM interrupted HBV entry and would therefore suppress HBV re-infection.

Article	Year
A rhodol-based fluorescent chemosensor for hydrazine and its application in live cell bioimaging. Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy\| PubMed ID: 28582724	2017
A rhodamine-triazole fluorescent chemodosimeter for Cu detection and its application in bioimaging. Luminescence : the journal of biological and chemical luminescence\| PubMed ID: 29327432	2018
A novel immortalized hepatocyte-like cell line (imHC) supports in vitro liver stage development of the human malarial parasite Plasmodium vivax. Malaria journal\| PubMed ID: 29370800	2018
A chromogenic and fluorogenic rhodol-based chemosensor for hydrazine detection and its application in live cell bioimaging. Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy\| PubMed ID: 29414570	2018
An Immortalized Hepatocyte-like Cell Line (imHC) Accommodated Complete Viral Lifecycle, Viral Persistence Form, cccDNA and Eventual Spreading of a Clinically-Isolated HBV. Viruses\| PubMed ID: 31623162	2019
Effective suppression of yellow head virus replication in Penaeus monodon hemocytes using constitutive expression vector for long-hairpin RNA (lhRNA). Journal of invertebrate pathology\| PubMed ID: 32663545	2020
High-content screening of Thai medicinal plants reveals Boesenbergia rotunda extract and its component Panduratin A as anti-SARS-CoV-2 agents. Scientific reports\| PubMed ID: 33203926	2020
Immortalized stem cell-derived hepatocyte-like cells: An alternative model for studying dengue pathogenesis and therapy. PLoS neglected tropical diseases\| PubMed ID: 33216752	2020
Anti-SARS-CoV-2 Activity of Extract and Its Major Component Andrographolide in Human Lung Epithelial Cells and Cytotoxicity Evaluation in Major Organ Cell Representatives. Journal of natural products\| PubMed ID: 33844528	2021
A Rhodamine-based Fluorescent Chemodosimeter for Au in Aqueous Solution and Living Cells. Journal of fluorescence\| PubMed ID: 34046770	2021
Curcumin inhibited hepatitis B viral entry through NTCP binding. Scientific reports\| PubMed ID: 34580340	2021

Yongyut Pewkliang

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