Impact of physicochemical parameters on in vitro assembly and disassembly kinetics of recombinant triple‐layered rotavirus‐like particles

Mellado, Maria Cândida M.; Mena, Jimmy A.; Lopes, António; Ramı́rez, Octavio T.; Carrondo, Manuel J.T.; Palomares, Laura A.; Alves, Paula M.

doi:10.1002/bit.22430

Cited by 28 publications

(24 citation statements)

References 59 publications

(80 reference statements)

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“…The 3D reconstruction also indicated a single significant population of DLPs was present in the EDTA-treated samples. Our findings are consistent with previous light scattering measurements that showed a complete conversion of TLPs to DLPs in vitro within 2 min in the presence of 2 mM EDTA [22]. …”

Section: Converting Tlps To Dlpssupporting

confidence: 93%

Molecular Surveillance of Viral Processes Using Silicon Nitride Membranes

et al. 2013

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Abstract:Here we present new applications for silicon nitride (SiN) membranes to evaluate biological processes. We determined that 50-nanometer thin films of SiN produced from silicon wafers were sufficiently durable to bind active rotavirus assemblies. A direct comparison of SiN microchips with conventional carbon support films indicated that SiN performs equivalent to the traditional substrate to prepare samples for Electron Microscopy (EM) imaging. Likewise, SiN films coated with Ni-NTA affinity layers concentrated rotavirus particles similarly to affinity-coated carbon films. However, affinity-coated SiN membranes outperformed glow-discharged conventional carbon films 5-fold as indicated by the number of viral particles quantified in EM images. In addition, we were able to recapitulate viral uncoating and transcription mechanisms directed onto the microchip surfaces. EM images of these processes revealed the production of RNA transcripts emerging from active rotavirus complexes. These results were confirmed by the functional incorporation of radiolabeled nucleotides into the nascent RNA transcripts. Collectively, we demonstrate new uses for SiN membranes to perform molecular surveillance on life processes in real-time. OPEN ACCESSMicromachines 2013, 4 91

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Section: Converting Tlps To Dlpssupporting

confidence: 93%

Molecular Surveillance of Viral Processes Using Silicon Nitride Membranes

et al. 2013

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“…Mellado et al [12] studied the disassembly (and reassembly) of a triple-layered rotavirus-like particle (tlRLP) into the double-layered one (dlRLP) by CZE. Such virus-like particles are important for the manufacture of vaccines because they closely mimic the viral antigen but are non-infectious as they lack the viral genome.…”

Section: Virusesmentioning

confidence: 99%

Recent developments in capillary and chip electrophoresis of bioparticles: Viruses, organelles, and cells

2011

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In appropriate aqueous buffer solutions, biological particles usually exhibit a particular electric surface charge due to exposed charged or chargeable functional groups (amino acid residues, acidic carbohydrate moieties, etc.). Consequently, these bioparticles can migrate in solution under the influence of an electric field allowing separation according to their electrophoretic mobilities or their pI values. Based on these properties, electromigration methods are of eminent interest for the characterization, separation, and detection of such particles. The present review discusses the research papers published between 2008 and 2010 dealing with isoelectric focusing and zone electrophoresis of viruses, organelles and microorganisms (bacteria and yeast cells) in the capillary and the chip format.

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“…HepG2 cell line) (Vicente et al, 2009a). The purity of rBac solutions, including single viral particles and aggregates, can be indirectly measured by flow cytometry (Jorio et al, 2006), capillary zone electrophoresis and dynamic light scattering, DLS (Konz et al, 2005;Mellado et al, 2009;Vicente et al, 2009a;Wright et al, 2005). Total DNA content is estimated using the PicoGreen dsDNA Assay kit; the integrity and morphology of rBac are monitored by TEM (Vicente et al, 2009a).…”

Section: Rbac As the Final Productmentioning

confidence: 99%