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SUMMARY With increasing resistance to anti-parasitic drugs, it has become more important to detect and recognize phenotypes of resistant isolates. Molecular methods of detecting resistant isolates are limited at present. Here, we introduce a microfluidic bioassay to measure phenotype using parameters of nematode locomotion. We illustrate the technique on larvae of an animal parasite Oesophagostomum dentatum. Parameters of sinusoidal motion such as propagation velocity, wavelength, wave amplitude, and oscillation frequency depended on the levamisole-sensitivity of the isolate of parasitic nematode. The levamisole-sensitive isolate (SENS) had a mean wave amplitude of 135 μ m, which was larger than 123 μ m of the levamisole-resistant isolate (LEVR). SENS had a mean wavelength of 373 μ m, which was less than 393 μ m of LEVR. The mean propagation velocity of SENS, 149 μ m s −1 , was similar to LEVR, 143 μ m s −1 . The propagation velocity of the isolates was inhibited by levamisole in a concentration-dependent manner above 0·5 μ m . The EC 50 for SENS was 3 μ m and the EC 50 for LEVR was 10 μ m . This microfluidic technology advances present-day nematode migration assays and provides a better quantification and increased drug sensitivity. It is anticipated that the bioassay will facilitate study of resistance to other anthelmintic drugs that affect locomotion.
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A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutations
The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mproinhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mproinhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as an approximation for Mproactivity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays identified substitutions which conferred strong nirmatrelvir resistance and others that compromised activity. On the other hand, N142A and the P132H mutation, carried by the Omicron variant, caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of MproE166R, and MproE166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mprothat may arise as Mproantivirals become more widely used.
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- Award ID(s):
- 2200138
- PAR ID:
- 10477872
- Editor(s):
- Liu, Wenshe
- Publisher / Repository:
- PLOS
- Date Published:
- Journal Name:
- PLOS Pathogens
- Volume:
- 19
- Issue:
- 8
- ISSN:
- 1553-7374
- Page Range / eLocation ID:
- e1011592
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1371/journal.ppat.1011592
