Identification of anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) oxysterol derivatives in vitro

Development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) are urgently needed to combat the coronavirus disease 2019 (COVID-19). Oxysterols, defined as oxidized derivatives of cholesterol, include endogenous (naturally occurring) cholesterol metabolites as well as semi-synthetic oxysterol derivatives. We have previously studied the use of semi-synthetic oxysterol derivatives as drug candidates for inhibition of cancer, fibrosis, and bone regeneration. In this study, we have screened a panel of naturally occurring and semi-synthetic oxysterol derivatives for anti-SARS-CoV-2 activity, using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy186 displayed antiviral activity comparable to natural oxysterols. In addition, related oxysterol analogues Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 μM and 99% at 15 μM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fall into a therapeutically relevant range (19 μM), based on the dose-dependent curve for antiviral activity in our cell culture infection assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 with disrupting the formation of double membrane vesicles (DMVs), intracellular membrane compartments associated with viral replication. Oxy210 also inhibited the replication of hepatitis C virus, another RNA virus whose replication is associated with DMVs, but not the replication of the DMV-independent hepatitis D virus. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk developing COVID-19.

Modified techniques in quantification of intracellular Listeria monocytogenes in vitro infection

The demand for reliable methods for the quantification of intracellular bacteria is growing. Among modern methods such as PCR and flow cytometry, traditional methods including colony forming unit assay and immune-fluorescence are still the two most commonly techniques worldwide. In colony forming unit assay, there are variations among publications, making data results inconsistent across studies. The aim of this paper is to evaluate available techniques and develop improved protocols for the quantification of intracellular Listeria monocytogenes (LM) in vitro infection assay. This study has suggested different uptake time for phagocytic and non-phagocytic cells. Specifically, uptake time was determined at 0.5 hour after infection for RAW264.7 macrophages and 2 hours for L929 fibroblast host cells. To efficiently remove extracellular bacteria during infection period, gentamicin at high and low concentrations was used during the infection assay. High concentration of gentamicin was used to kill extracellular bacteria while low concentration of gentamicin was used to prevent secondary infection of host cells during the infection period. To obtain a more accurate number of alive LM from a large scale experiment, phosphate-buffered saline/PBS should be used rather than mili-Q (mQ) water to lyse the host cell as mQ water can kill additional bacteria unexpectedly. In immune-fluorescence, LM can be visualized by using either the LM expressing green fluorescence protein (GFP) or antibody against LM. To observed GFP signal, cells should be fixed with paraformaldehyde as methanol will rapidly dim the GFP signal. Findings from this study will benefit researchers engaged in both basic cell biology and infectious diseases.

Evaluation of Anti-Mycobacterial Compounds in a Silkworm Infection Model with Mycobacteroides abscessus

Among four mycobacteria, Mycobacterium avium, M. intracellulare, M. bovis BCG and Mycobacteroides (My.) abscessus, we established a silkworm infection assay with My. abscessus. When silkworms (fifth-instar larvae, n = 5) were infected through the hemolymph with My. abscessus (7.5 × 107 CFU/larva) and bred at 37 °C, they all died around 40 h after injection. Under the conditions, clarithromycin and amikacin, clinically used antimicrobial agents, exhibited therapeutic effects in a dose-dependent manner. Furthermore, five kinds of microbial compounds, lariatin A, nosiheptide, ohmyungsamycins A and B, quinomycin and steffimycin, screened in an in vitro assay to observe anti-My. abscessus activity from 400 microbial products were evaluated in this silkworm infection assay. Lariatin A and nosiheptide exhibited therapeutic efficacy. The silkworm infection model with My. abscessus is useful to screen for therapeutically effective anti-My. abscessus antibiotics.

An Efficient in vitro Assay to Predict Resistance and Pathogenicity in the Fusarium Head Blight-Hordeum Vulgare Pathosystem

Background: Barley (Hordeum vulgare L.) worldwide is affected seriously by Fusarium Head Blight (FHB) disease caused by several Fusarium species. In vitro assays permitting for easy, efficient and reliable prediction of the head blight reaction in the whole plant should be investigated. Objective and Methods: The in vitro ability of 16 fungal isolates of four FHB species to confer disease on individual plant organs was evaluated using a coleoptile infection assay. Four quantitative components (Seed Germination (SG), Coleoptile Length (CL), Coleoptile Weight (CW) and Root Weight (RW)) were analyzed in two widely cultivated barley cultivars, Arabi Aswad (AS) and Arabi Abiad (AB), with known quantitative resistance. Results: Differences in inoculated pathogenicity and resistance treatments were observed on young plant parts relative to water controls, indicating that these FHB species were found to be suitable for the differential expression of all tested quantitative components. There was a wide variation in pathogenicity among the 16 FHB isolates and susceptibility among AS and AB. The 16 FHB isolates can be separated into the first group with larger number of isolates, upon infection with which AB really was more susceptible to FHB infection than AS, and isolates of the second group with leaser number of isolates for which AS and AB react was the opposite. On AB, rather susceptible, inoculation with FHB species resulted in significantly less SG, CL, CW and RW, compared with AS, which showed a greater resistance. The very good resistance of AS was confirmed by the measurements of quantitative resistance components described in this study. When infected with FHB isolates, all indicators of a more susceptible cultivar seemed to be 10-20% less than those of a resistant cultivar. Moreover, the values of all analyzed components were significantly correlated with the data of pathogenic indices generated in vitro, and under controlled and field conditions with a large diversity depending on AS and AB. Conclusion: Appropriate in vitro conditions were determined for the coleoptile infection assay to maximize differences in disease reactions components among FHB isolates and the two barley cultivars. Results suggest that all measured components predict resistance and pathogenicity occurring at the earliest and latest barley development stages during FHB infection. Our data also highlighted, for the first time, the utility of CW and RW for the determination of resistance and pathogenicity in the FHB-barley pathosystem. The coleoptile infection test was confirmed to be adequate to in vitro, growth chamber and field data by the presence of the first group, which prevailed in all other tests generated under different experimental conditions. The in vitro coleoptile infection assay may offer a real possibility of simple, rapid and reliable screening of resistance in barley cultivars and pathogenicity of FHB species.