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  1. Abstract

    SARS-CoV-2 is an RNA enveloped virus responsible for the COVID-19 pandemic that conducted in 6 million deaths worldwide so far. SARS-CoV-2 particles are mainly composed of the 4 main structural proteins M, N, E and S to form 100 nm diameter viral particles. Based on productive assays, we propose an optimal transfected plasmid ratio mimicking the viral RNA ratio in infected cells. This allows SARS-CoV-2 Virus-Like Particle (VLPs) formation composed of the viral structural proteins M, N, E and mature S. Furthermore, fluorescent or photoconvertible VLPs were generated by adding a fluorescent protein tag on N or M mixing with unlabeled viral proteins and characterized by western blots, atomic force microscopy coupled to fluorescence and immuno-spotting. Thanks to live fluorescence and super-resolution microscopies, we quantified VLPs size and concentration. SARS-CoV-2 VLPs present a diameter of 110 and 140 nm respectively for MNE-VLPs and MNES-VLPs with a concentration of 10e12 VLP/ml. In this condition, we were able to establish the incorporation of the Spike in the fluorescent VLPs. Finally, the Spike functionality was assessed by monitoring fluorescent MNES-VLPs docking and internalization in human pulmonary cells expressing or not the receptor hACE2. Results show a preferential maturation of S on N(GFP) labeled VLPsmore »and an hACE2-dependent VLP internalization and a potential fusion in host cells. This work provides new insights on the use of non-fluorescent and fluorescent VLPs to study and visualize the SARS-CoV-2 viral life cycle in a safe environment (BSL-2 instead of BSL-3). Moreover, optimized SARS-CoV-2 VLP production can be further adapted to vaccine design strategies.

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  2. Formation of vitreous ice during rapid compression of water at room temperature is important for biology and the study of biological systems. Here, we show that Raman spectra of rapidly compressed water at greater than 1 GPa at room temperature exhibits the signature of high-density amorphous ice, whereas the X-ray diffraction (XRD) pattern is dominated by crystalline ice VI. To resolve this apparent contradiction, we used molecular dynamics simulations to calculate full vibrational spectra and diffraction patterns of mixtures of vitreous ice and ice VI, including embedded interfaces between the two phases. We show quantitatively that Raman spectra, which probe the local polarizability with respect to atomic displacements, are dominated by the vitreous phase, whereas a small amount of the crystalline component is readily apparent by XRD. The results of our combined experimental and theoretical studies have implications for detecting vitreous phases of water, survival of biological systems under extreme conditions, and biological imaging. The results provide additional insight into the stable and metastable phases of H 2 O as a function of pressure and temperature, as well as of other materials undergoing pressure-induced amorphization and other metastable transitions.
    Free, publicly-accessible full text available July 5, 2023
  3. Abstract

    SARS-CoV-2 virus is the causative agent of COVID-19. Here we demonstrate that non-infectious SARS-CoV-2 virus like particles (VLPs) can be assembled by co-expressing the viral proteins S, M and E in mammalian cells. The assembled SARS-CoV-2 VLPs possess S protein spikes on particle exterior, making them ideal for vaccine development. The particles range in shape from spherical to elongated with a characteristic size of 129 ± 32 nm. We further show that SARS-CoV-2 VLPs dried in ambient conditions can retain their structural integrity upon repeated scans with Atomic Force Microscopy up to a peak force of 1 nN.