996 research outputs found
Bioorthogonal Strategy for Bioprocessing of Specific-Site-Functionalized Enveloped Influenza-Virus-Like Particles
Virus-like particles (VLPs) constitute a promising platform in vaccine development and targeted drug delivery. To date, most applications use simple nonenveloped VLPs as human papillomavirus or hepatitis B vaccines, even though the envelope is known to be critical to retain the native protein folding and biological function. Here, we present tagged enveloped VLPs (TagE-VLPs) as a valuable strategy for the downstream processing and monitoring of the in vivo production of specific-site-functionalized enveloped influenza VLPs. This two-step procedure allows bioorthogonal functionalization of azide-tagged nascent influenza type A hemagglutinin proteins in the envelope of VLPs through a strain-promoted [3 + 2] alkyne-azide cycloaddition reaction. Importantly, labeling does not influence VLP production and allows for construction of functionalized VLPs without deleterious effects on their biological function. Refined discrimination and separation between VLP and baculovirus, the major impurity of the process, is achieved when this technique is combined with flow cytometry analysis, as demonstrated by atomic force microscopy. TagE-VLPs is a versatile tool broadly applicable to the production, monitoring, and purification of functionalized enveloped VLPs for vaccine design trial runs, targeted drug delivery, and molecular imaging.The authors acknowledge funding from the European Union (EDUFLUVAC project FP7-HEALTH-2013-INNOVATION), the Fundação para a CiĂȘncia e Tecnologia (FCT, Portugal; project HIVERA/0002/2013 and FCT Investigator to G.J.L.B.), EPSRC (to G.J.L.B.), the European Commission, Marie SkĆodowska-Curie Actions (MSCA), and RISE project grant 644167. S. B. C., J. M. F., F. M., and D. G. acknowledge FCT for fellowships SFRH/BD/52302/2013, SFRH/BD/70423/2010, SFRH/BD/70139/2010, and SFRH/BPD/73500/2010, respectively. The authors acknowledge Ricardo Silva for all his help in fluorescence analysis implementation and fruitful discussions. The authors also acknowledge PatrĂcia Gomes-Alves for her help for mass spectrometry analysis. Mass spectrometry data was obtained by the Mass Spectrometry Unit (UniMS), ITQB/iBET, Oeiras, Portugal. G. J. L. B. is a Royal Society University Research Fellow and the recipient of a European Research Council Starting Grant (TagIt)
The role of c-Met and VEGFR2 in glioblastoma resistance to bevacizumab
Dismal prognosis of glioblastoma (GBM) prompts for the identification of response predictors and therapeutic resistance mechanisms of current therapies. The authors investigated the impact of c-Met, HGF, VEGFR2 expression and microvessel density (MVD) in GBM patients submitted to second-line chemotherapy with bevacizumab. Immunohistochemical expression of c-Met, HGF, VEGFR2, and MVD was assessed in tumor specimens of GBM patients treated with bevacizumab, after progression under temozolomide. Survival analysis was evaluated according to the expression of the aforementioned biomarkers. c-Met overexpression was associated with a time-to-progression (TTP) after bevacizumab of 3 months (95% CI, 1.5â4.5) compared with a TTP of 7 months (95% CI, 4.6â9.4) in patients with low or no expression of c-Met (p = 0.05). VEGFR2 expression was associated with a TTP after bevacizumab of 3 months (95% CI, 1.8â4.2) compared with a TTP of 7 months (95% CI, 5.7â8.3) in patients with no tumoral expression of VEGFR2 (p = 0.009). Concomitant c-Met/VEGFR2 overexpression was associated with worse overall survival (13 months) compared with concomitant c-Met/VEGFR2 negative expression (19 months; p = 0.025). Our data support the hypothesis that c-Met and VEGFR2 overexpression have a role in the development of glioblastoma early resistance and might predict poorer responses to anti-angiogenic therapies.This work was financed by FEDERâFundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020âOperacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT-Fundação para a CiĂȘncia e a Tecnologia/MinistĂ©rio da CiĂȘncia, Tecnologia e Inovação in the framework of the project âInstitute for Research and Innovation in Health Sciencesâ (POCI-01-0145-FEDER-007274). Additional funding by the European Regional Development Fund (ERDF), COMPETE2020 and Portuguese national funds via FCT, under project POCI-01-0145-FEDER-016390: CANCEL STEM and the project POCI-01-0145-FEDER-031438 (PDTC/MED_ONC/31438/2017)
A shadowing problem in the detection of overlapping communities: lifting the resolution limit through a cascading procedure
Community detection is the process of assigning nodes and links in
significant communities (e.g. clusters, function modules) and its development
has led to a better understanding of complex networks. When applied to sizable
networks, we argue that most detection algorithms correctly identify prominent
communities, but fail to do so across multiple scales. As a result, a
significant fraction of the network is left uncharted. We show that this
problem stems from larger or denser communities overshadowing smaller or
sparser ones, and that this effect accounts for most of the undetected
communities and unassigned links. We propose a generic cascading approach to
community detection that circumvents the problem. Using real and artificial
network datasets with three widely used community detection algorithms, we show
how a simple cascading procedure allows for the detection of the missing
communities. This work highlights a new detection limit of community structure,
and we hope that our approach can inspire better community detection
algorithms.Comment: 14 pages, 12 figures + supporting information (5 pages, 6 tables, 3
figures
Intercomparison of the northern hemisphere winter mid-latitude atmospheric variability of the IPCC models
We compare, for the overlapping time frame 1962-2000, the estimate of the
northern hemisphere (NH) mid-latitude winter atmospheric variability within the
XX century simulations of 17 global climate models (GCMs) included in the
IPCC-4AR with the NCEP and ECMWF reanalyses. We compute the Hayashi spectra of
the 500hPa geopotential height fields and introduce an integral measure of the
variability observed in the NH on different spectral sub-domains. Only two
high-resolution GCMs have a good agreement with reanalyses. Large biases, in
most cases larger than 20%, are found between the wave climatologies of most
GCMs and the reanalyses, with a relative span of around 50%. The travelling
baroclinic waves are usually overestimated, while the planetary waves are
usually underestimated, in agreement with previous studies performed on global
weather forecasting models. When comparing the results of various versions of
similar GCMs, it is clear that in some cases the vertical resolution of the
atmosphere and, somewhat unexpectedly, of the adopted ocean model seem to be
critical in determining the agreement with the reanalyses. The GCMs ensemble is
biased with respect to the reanalyses but is comparable to the best 5 GCMs.
This study suggests serious caveats with respect to the ability of most of the
presently available GCMs in representing the statistics of the global scale
atmospheric dynamics of the present climate and, a fortiori, in the perspective
of modelling climate change.Comment: 39 pages, 8 figures, 2 table
Large emissions from floodplain trees close the Amazon methane budget
Wetlands are the largest global source of atmospheric methane (CH4), a potent greenhouse gas. However, methane emission inventories from the Amazon floodplain, the largest natural geographic source of CH4 in the tropics, consistently underestimate the atmospheric burden of CH4 determined via remote sensing and inversion modelling, pointing to a major gap in our understanding of the contribution of these ecosystems to CH4 emissions. Here we report CH4 fluxes from the stems of 2,357 individual Amazonian floodplain trees from 13 locations across the central Amazon basin. We find that escape of soil gas through wetland trees is the dominant source of regional CH4 emissions. Methane fluxes from Amazon tree stems were up to 200 times larger than emissions reported for temperate wet forests6 and tropical peat swamp forests, representing the largest non-ebullitive wetland fluxes observed. Emissions from trees had an average stable carbon isotope value (ÎŽ13C) of â66.2â±â6.4 per mil, consistent with a soil biogenic origin. We estimate that floodplain trees emit 15.1â±â1.8 to 21.2â±â2.5 teragrams of CH4 a year, in addition to the 20.5â±â5.3 teragrams a year emitted regionally from other sources. Furthermore, we provide a âtop-downâ regional estimate of CH4 emissions of 42.7â±â5.6 teragrams of CH4 a year for the Amazon basin, based on regular vertical lower-troposphere CH4 profiles covering the period 2010â2013. We find close agreement between our âtop-downâ and combined âbottom-upâ estimates, indicating that large CH4 emissions from trees adapted to permanent or seasonal inundation can account for the emission source that is required to close the Amazon CH4 budget. Our findings demonstrate the importance of tree stem surfaces in mediating approximately half of all wetland CH4 emissions in the Amazon floodplain, a region that represents up to one-third of the global wetland CH4 source when trees are combined with other emission sources
Optimal interdependence between networks for the evolution of cooperation
Recent research has identified interactions between networks as crucial for the outcome of evolutionary
games taking place on them. While the consensus is that interdependence does promote cooperation by
means of organizational complexity and enhanced reciprocity that is out of reach on isolated networks, we
here address the question just how much interdependence there should be. Intuitively, one might assume
the more the better. However, we show that in fact only an intermediate density of sufficiently strong
interactions between networks warrants an optimal resolution of social dilemmas. This is due to an intricate
interplay between the heterogeneity that causes an asymmetric strategy flow because of the additional links
between the networks, and the independent formation of cooperative patterns on each individual network.
Presented results are robust to variations of the strategy updating rule, the topology of interdependent
networks, and the governing social dilemma, thus suggesting a high degree of universality
A comparison of methods for purification and concentration of norovirus GII-4 capsid virus-like particles
Noroviruses (NoVs) are one of the leading causes of acute gastroenteritis worldwide. NoV GII-4 VP1 protein was expressed in a recombinant baculovirus system using Sf9 insect cells. Several methods for purification and concentration of virus-like particles (VLPs) were evaluated. Electron microscopy (EM) and histo-blood group antigen (HBGA) binding assays showed that repeated sucrose gradient purification followed by ultrafiltration resulted in intact VLPs with excellent binding to H type 3 antigens. VLPs were stable for at least 12 months at 4°C, and up to 7 days at ambient temperature. These findings indicate that this method yielded stable and high-quality VLPs
On the Effect of Thermodynamic Equilibrium on the Assembly Efficiency of Complex Multi-Layered Virus-Like Particles (VLP): the Case of Rotavirus VLP
Previous studies have reported the production of malformed virus-like-particles (VLP) in recombinant host systems. Here we computationally investigate the case of a large triple-layered rotavirus VLP (RLP). In vitro assembly, disassembly and reassembly data provides strong evidence of microscopic reversibility of RLP assembly. Light scattering experimental data also evidences a slow and reversible assembly untypical of kinetic traps, thus further strengthening the fidelity of a thermodynamically controlled assembly. In silico analysis further reveals that under favourable conditions particles distribution is dominated by structural subunits and completely built icosahedra, while other intermediates are present only at residual concentrations. Except for harshly unfavourable conditions, assembly yield is maximised when proteins are provided in the same VLP protein mass composition. The assembly yield decreases abruptly due to thermodynamic equilibrium when the VLP protein mass composition is not obeyed. The latter effect is more pronounced the higher the Gibbs free energy of subunit association is and the more complex the particle is. Overall this study shows that the correct formation of complex multi-layered VLPs is restricted to a narrow range of association energies and protein concentrations, thus the choice of the host system is critical for successful assembly. Likewise, the dynamic control of intracellular protein expression rates becomes very important to minimize wasted proteins
Intra and interobserver reliability of the interpretation of high-resolution computed tomography on the lungs of premature infants
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