Cell cycle analysis examined using flow cytometry on HT-29 cells after 72 h treatment.

<p>(A) Cells treated with (a) DMSO at the final concentration of 0.1%. (b) FKB at a concentration of 12.5 (3.55 μg/mL), (c) 25 (7.1 μg/mL), (d) 50 μM (14.2 μg/mL), and (e) Percentage of cell cycle distribution in different phases. (B) Cells treated with (a) DMSO at the final concentration of 0.1%. (b) APN at 12.5 (3.37 μg/mL), (c) 25 (6.75 μg/mL), (d) 50 μM (13.5 μg/mL) concentrations, and (e) Percentage of cell cycle distribution in different phases. G0/G1, G2+M, and S are cell phases, respectively; subG0/G1 refers to cell death due to DNA fragmentation. Data are expressed as Mean±SD of three independent experiments, *p<0.001, ns: non-significant compared to the normal control.</p

DNA fragmentation analysed in 1% agarose gel after 72 h incubation with different concentration of either FKB or APN.

<p>MW: DNA marker; DC: DMSO treated control; all concentrations are in μM.</p

Levels of MDM2 and p53 proteins expressed in HT-29 cells.

<p>(A) Level of proteins in cells treated with (a) crude hexane (IC₂₅: 10.52, IC₅₀: 21.05, and IC₇₅:42.1 μg/mL) and chloroform (IC₂₅: 9.5, IC₅₀: 19.09, and IC₇₅:38.18 μg/mL) extracts. (b) 25 μM (7.1 μg/ mL) of FKB at different time interval. (c) 25 μM (6.75 μg/mL) of APN at different time interval. (B) Level of MDM2 and p53 protein expression quantified from western blotting analysis using Bio-rad Image Lab software in HT 29 cells treated with (a) Hexane and chloroform extracts (b) FKB, and (c) APN. Data are expressed as Mean±SD; ns: non-significant; *p<0.05; **p<0.01; ***p<0.01; ns: non-significant compared to the DMSO control. DC: DMSO used as negative control at a final concentration of 0.1%.</p

DataSheet_1_Erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin inhibit cytopathic effect, papain-like protease, and MPRO enzymes of SARS-CoV-2.pdf

BackgroundAlthough tremendous success has been achieved in the development and deployment of effective COVID-19 vaccines, developing effective therapeutics for the treatment of those who do come down with the disease has been with limited success. To repurpose existing drugs for COVID-19, we previously showed, qualitatively, that erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin inhibit SARS-COV-2-induced cytopathic effect (CPE) in Vero cells.AimThis study aimed to quantitatively explore the inhibition of SARS-CoV-2-induced CPE by erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin and to determine the effect of these drugs on SARS-CoV-2 papain-like protease and 3CL protease (MPRO) enzymes.MethodsNeutral red (3-amino-7-dimethylamino-2-methyl-phenazine hydrochloride) cell viability assay was used to quantify CPE after infecting pre-treated Vero cells with clinical SARS-Cov-2 isolates. Furthermore, SensoLyte® 520 SARS-CoV-2 papain-like protease and SensoLyte® 520 SARS-CoV-2 MPRO activity assay kits were used to evaluate the inhibitory activity of the drugs on the respective enzymes.ResultsErythromycin, retapamulin, pyridoxine, folic acid, and ivermectin dose-dependently inhibit SARS-CoV-2-induced CPE in Vero cells, with inhibitory concentration-50 (IC50) values of 3.27 µM, 4.23 µM, 9.29 µM, 3.19 µM, and 84.31 µM, respectively. Furthermore, erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin dose-dependently inhibited SARS-CoV-2 papain-like protease with IC50 values of 0.94 µM, 0.88 µM, 1.14 µM, 1.07 µM, and 1.51 µM, respectively, and inhibited the main protease (MPRO) with IC50 values of 1.35 µM, 1.25 µM, 7.36 µM, 1.15 µM, and 2.44 µM, respectively.ConclusionThe IC50 for all the drugs, except ivermectin, was at the clinically achievable plasma concentration in humans, which supports a possible role for the drugs in the management of COVID-19. The lack of inhibition of CPE by ivermectin at clinical concentrations could be part of the explanation for its lack of effectiveness in clinical trials.</p

Table_1_Effects of Newcastle Disease Virus Infection on Chicken Intestinal Intraepithelial Natural Killer Cells.DOCX

<p>The intestinal intraepithelial natural killer cells (IEL-NK) are among the earliest effectors of antiviral immunity in chicken. Unfortunately, their role during Newcastle disease virus (NDV) infection remains obscure. Previous study has reported the development of a monoclonal antibody (mAb) known as 28-4, which is specifically directed against the CD3⁻ IEL-NK cells. In the present study, we used this mAb to investigate the effects of velogenic and lentogenic NDV infection on avian IEL-NK cells. Our findings revealed that chickens infected with velogenic NDV strains have a reduced population of purified CD3⁻/28-4⁺ IEL-NK cells as determined by flow cytometry. Furthermore, the CD3⁻/28-4⁺ IEL-NK cells from chicken infected with velogenic NDV strains were shown to have a downregulated expression of activating receptors (CD69 and B-Lec), effector peptide (NK-LYSIN), and IFN gamma. On the contrary, the expression of the inhibitory receptor (B-NK) and bifunctional receptor (CHIR-AB1) were upregulated on these purified CD3⁻/28-4⁺ IEL-NK cells following velogenic NDV infection. Meanwhile, the lentogenic NDV demonstrated insignificant effects on both the total population of CD3⁻/28-4⁺ IEL-NK cells and the expression of their surface receptors. In addition, using real-time PCR and transmission electron microscopy, we showed that CD3⁻/28-4⁺ IEL-NK cells were susceptible to velogenic but not lentogenic NDV infection. These findings put together demonstrate the ability of different strains of NDV to manipulate the activating and inhibitory receptors of CD3⁻/28-4⁺ IEL-NK cells following infection. Further studies are, however, required to ascertain the functional importance of these findings during virulent or avirulent NDV infection.</p