378 research outputs found
Enhanced borohydride oxidation kinetics at gold-rare earth alloys
No full textsponsorship: The authors would like to thank the Ministry of Education, Science and Technological Development of the Republic of Serbia (contract no. 451-03-68/2020-14/200146). The authors would also like to thank the Portuguese Foundation for Science and Technology (FCT, Portugal) for a research grant within project UID/CTM/04540/2013 (G. Backovic), for contract IST-ID/156/2018 (B. Sljukic), and for a research contract in the scope of programmatic funding UIDP/04540/2020 (D.M.F. Santos). (Ministry of Education, Science and Technological Development of the Republic of Serbia|451-03-68/2020-14/200146, Portuguese Foundation for Science and Technology (FCT, Portugal)|UID/CTM/04540/2013, Portuguese Foundation for Science and Technology (FCT, Portugal)|IST-ID/156/2018, Portuguese Foundation for Science and Technology (FCT, Portugal)|UIDP/04540/2020)status: Publishe
Capsid Assembly and Single Stranded DNA Genome Formation of Adeno-Associated Virus Type2 in Yeast Cells
No full textSaccharomyces cerevisiae has provided an array of genetic tools to study unknown aspects
of viral life cycles, supporting replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses). It also provides means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus) and as such represents a useful tool for vaccine development. To extend the utility of the S. cerevisiae expression system, we expressed AAV2 structural and nonstructural proteins in yeast cells, using both authentic AAV2 and heterologous yeast promoters. For the first time, we described the assembly of AAV2 virus-like particles from yeast-expressed AAV2 structural proteins. To do this we used AAV p40 promoter, whose activity in yeast cells resembled the one of yeast glycolytic promoters, resulting in the synthesis of the most abundant capsid protein VP3 when transformed yeast cells were
grown on glucose as a carbon source. The expression of other two VPs was induced from yeast, galactose inducible pGal1 promoter. Simultaneous production of all three VPs was achieved by growing the yeast cells in the medium containing both glucose and galactose, while their relative production levels were further optimized by varying amounts of each
carbon source in the induction medium, followed by the fine tuning of the induction time.
Moreover, we investigated the ability of the yeast Saccharomyces cerevisiae to carry out
the replication of a recombinant rAAV2. When a plasmid harboring the rAAV2 genome in
which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in
yeast with a plasmid expressing Rep68 from constitutive yeast promoter pADH, a
significant number of URA3+ clones were scored (more than 30-fold over controls).
Molecular analysis of low molecular weight DNA revealed that the single stranded DNA is
formed, in Rep68 and ITR dependent manner, and that the plasmid is entirely replicated.
The ss DNA contained the ITRs, URA3 gene and also vector sequences suggesting that ss
rAAV genomes were not obtained by the canonical AAV replication mechanism.
These results could open new prospects for using yeast cell in two ways: (i) as a model
system for studying viral and cellular factors involved in AAV2 capsid assembly and
packaging of rAAV ss genomes; and (ii) as a novel cell factory for developing superior
recombinant rAAV production technologies
Capsid protein expression and adeno-associated virus like particles assembly in Saccharomyces cerevisiae.
No full textThe budding yeast Saccharomyces cerevisiae supports replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses) and has provided means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus). We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid. In this work, we have investigated the possibility to assemble AAV capsids in yeast.To do this, at least two out of three AAV structural proteins, VP1 and VP3, have to be simultaneously expressed in yeast cells and their intracellular stoichiometry has to resemble the one found in the particles derived from mammalian or insect cells. This was achieved by stable co-transformation of yeast cells with two plasmids, one expressing VP3 from its natural p40 promoter and the other one primarily expressing VP1 from a modified AAV2 Cap gene under the control of the inducible yeast promoter Gal1. Among various induction strategies we tested, the best one to yield the appropriate VP1:VP3 ratio was 4.5 hour induction in the medium containing 0.5\% glucose and 5\% galactose. Following such induction, AAV virus like particles (VLPs) were isolated from yeast by two step ultracentrifugation procedure. The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells.Taken together, the results show for the first time that yeast can be used to assemble AAV capsid and, therefore, as a genetic system to identify novel cellular factors involved in AAV biology
Capsid protein expression and adeno-associated virus like particles assembly in <it>Saccharomyces cerevisiae</it>
No full textAbstract Background The budding yeast Saccharomyces cerevisiae supports replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses) and has provided means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus). We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid. In this work, we have investigated the possibility to assemble AAV capsids in yeast. Results To do this, at least two out of three AAV structural proteins, VP1 and VP3, have to be simultaneously expressed in yeast cells and their intracellular stoichiometry has to resemble the one found in the particles derived from mammalian or insect cells. This was achieved by stable co-transformation of yeast cells with two plasmids, one expressing VP3 from its natural p40 promoter and the other one primarily expressing VP1 from a modified AAV2 Cap gene under the control of the inducible yeast promoter Gal1. Among various induction strategies we tested, the best one to yield the appropriate VP1:VP3 ratio was 4.5 hour induction in the medium containing 0.5% glucose and 5% galactose. Following such induction, AAV virus like particles (VLPs) were isolated from yeast by two step ultracentrifugation procedure. The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells. Conclusions Taken together, the results show for the first time that yeast can be used to assemble AAV capsid and, therefore, as a genetic system to identify novel cellular factors involved in AAV biology.</p
Solar γ rays as a complementary probe of dark matter
No full textWe show that observations of solar γ rays offer a novel probe of dark matter in scenarios where interactions with the visible sector proceed via a long-lived mediator. As a proof of principle, we demonstrate that there exists a class of models which yield solar γ-ray fluxes observable with the next generation of γ-ray telescopes, while being allowed by a variety of current experimental constraints. The parameter space allowed by big bang nucleosynthesis and beam dump experiments naturally leads to mediator lifetimes sufficient to produce observable solar γ-ray signals. The model allows for solar γ-ray fluxes up to orders of magnitude larger compared to dwarf spheroidal galaxies, without reaching equilibrium between dark matter annihilation and capture rate. Our results suggest that solar γ-ray observations are complementary, and in some cases superior, to existing and future dark matter detection efforts
A determination of electroweak parameters at HERA
No full textUsing the deep inelastic e(+) p and e(-) p charged and neutral current scattering cross sections previously published, a combined electroweak and QCD analysis is performed to determine electroweak parameters accounting for their correlation with parton distributions. The data used have been collected by the HI experiment in 1994-2000 and correspond to an integrated luminosity of 117.2 pb(-1). A measurement is obtained of the W propagator mass in charged current ep scattering. The weak mixing angle sin(2)theta w is determined in the on-mass-shell renormalisation scheme. A first measurement at HERA is made of the light quark weak couplings to the Z(0) boson and a possible contribution of right-handed isospin components to the weak couplings is investigated
Probing dark matter long-lived mediators with solar γ rays
No full textWe show that solar γ-ray observations can provide a complementary probe of Dark Matter in scenarios where the interactions with the Standard Model proceed via long-lived mediators. For illustration we consider a simplified model which provides solar γ-ray fluxes observable with the next generation γ-ray telescopes, while complying with the existing experimental constraints. Our results suggest that solar γ-ray fluxes can be orders of magnitude larger than the ones from the Galactic center, while being subject to low backgrounds
Diffractive deep-inelastic scattering with a leading proton at HERA
No full textThe cross section for the diffractive deep-inelastic scattering process ep->eXp is measured, with the leading final state proton detected in the H1 Forward Proton Spectrometer. The data analysed cover the range xIP<0.1 in fractional proton longitudinal momentum loss, 0.08
Measurement and QCD analysis of the diffractive deep-inelastic scattering cross section at HERA
No full textA detailed analysis is presented of the diffractive deep-inelastic scattering process ep -> eXY, where Y is a proton or a low mass proton excitation carrying a fraction 1 - x(IP) > 0.95 of the incident proton longitudinal momentum and the squared four-momentum transfer at the proton vertex satisfie
Measurement of event shape variables in deep-inelastic scattering at HERA
No full textDeep-inelastic ep scattering data taken with the H1 detector at HERA and corresponding to an integrated luminosity of 106 pb(-1) are used to study the differential distributions of event shape variables. These include thrust, jet broadening, jet mass and the C-parameter. The four-momentum transfer Q is taken to be the relevant energy scale and ranges between 14 GeV and 200 GeV. The event shape distributions are compared with perturbative QCD predictions, which include resummed contributions and analytical power law corrections, the latter accounting for non-perturbative hadronisation effects. The data clearly exhibit the running of the strong coupling alpha(s)(Q) and are consistent with a universal power correction parameter alpha(0) for all event shape variables. A combined QCD fit using all event shape variables yields alpha(s)(m(Z)) = 0.1198 +/- 0.0013 (+0.0056)(-0.0043) and alpha(0) = 0.476 +/- 0.008 (+0.018)(-0.059)
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