Concepts for the Production of Viruses and Viral Vectors in Cell Cultures

The industrial-scale manufacturing of viruses or virus-like particles in cell culture is necessary for gene therapy and the treatment of cancer with oncolytic viruses. Complex multistep processes are required in both cases, but the low virus titers in batch cultures and the temperature sensitivity of the virus particles limit the production scale. To meet commercial and regulatory requirements, each process must be scalable and reproducible and must yield high virus titers. These requirements are met by establishing a cell culture process that matches the properties of the virus/host-cell system and by using serum-free cell culture medium. This chapter focuses on two case studies to consider the different aspects of process design, such as the reactor configuration and operational mode: the continuous production of retroviral pseudotype vectors in a retroviral packaging cell line and the production of oncolytic measles virus vectors for cancer therapy

Estudo da regeneração florestal de uma área de caíva com extração de erva-mate e pastejo animal no Planalto Norte Catarinense.

Caívas são remanescentes de florestas nativas com diferentes níveis de adensamento florestal, cujos estratos herbáceos são compostos por pastagens nativas, extensivamente pastejadas. Essas áreas de caívas, ainda que em diferentes estágios sucessionais, são importantes referências ambientais para diversas espécies da fauna e da flora local e ocupam, aproximadamente 13% da área do Planalto Norte Catarinense. Por serem áreas antropizadas, sua manutenção depende, em grande parte, do potencial de regeneração das espécies florestais presentes. Este trabalho teve por objetivo realizar o levantamento fitossociológico da regeneração de uma caíva, com área total de 10 ha, que vem sendo utilizada para extração de erva-mate nativa e pastejo animal há mais de 60 anos. O levantamento foi realizado em novembro e dezembro de 2010, em uma área experimental de 4.320 m2 demarcada dentro da caíva, onde foram alocadas 27 parcelas amostrais de 144m² cada. Todos os indivíduos arbustivos inferiores a 50 cm de altura, presentes nas parcelas, foram medidos e identificados. A identificação taxonômica foi realizada em campo com o apoio de um mateiro com experiência. A ordenação das famílias e gêneros foi baseada no APG III e a nomenclatura das espécies foi verificada nos arquivos on-line de Missouri Botanical Garden. Para análise fitossociológica foram realizadas as análises de frequência e dominância, absoluta e relativa. As plântulas foram agrupadas por altura, nos níveis de 0-10; 11-20; 21-40 e >41 cm. Na ordenação por altura, a maior concentração de plântulas ocorreu no extrato de 21-40 cm, sendo que foram encontradas 52% das plântulas neste extrato. Dentre as 1.641 plântulas amostradas foram identificadas 31 espécies, 19 famílias e 29 gêneros botânicos. Das espécies com maior densidade destacaram-se Zanthoxylum sp. (mamica de cadela), Allophylus edulis (vacum), Myrcia sp. (guamirim), Ilex microdonta Reissek (congonha), Mosiera prismatica (cerninho). Com relação à freqüência, as espécies que mais se destacaram foram: Zanthoxylum sp. (mamica de cadela), Allophylus edulis (vacum), Myrcia sp. (guamirim), Ilex microdonta Reissek (congonha), Ocotea sp. (canela). A quantidade e diversidade de espécies das plântulas levantadas na área amostral indicam que o processo de regeneração das áreas de caíva é intenso e atende a demanda para manutenção das características das caívas da região

The Cdc31p-binding protein Kar1p is a component of the half bridge of the yeast spindle pole body

KAR1 has been identified as an essential gene which is involved in karyogamy of mating yeast cells and in spindle pole body duplication of mitotic cells (Rose, M. D., and G. R. Fink. 1987. Cell. 48:1047-1060). We investigated the cell cycle-dependent localization of the Kar1 protein (Kar1p) and its interaction with other SPB components. Kar1p is associated with the spindle pole body during the entire cell cycle of yeast. Immunoelectron microscopic studies with anti-Kar1p antibodies or with the monoclonal antibody 12CA5 using an epitope-tagged, functional Kar1p revealed that Kar1p is associated with the half bridge or the bridge of the spindle pole body. Cdc31p, a Ca(2+)-binding protein, was previously identified as the first component of the half bridge of the spindle pole body (Spang, A., I. Courtney, U. Fackler, M. Matzner, and E. Schiebel. 1993. J. Cell Biol. 123:405-416). Using an in vitro assay we demonstrate that Cdc31p specifically interacts with a short sequence within the carboxyl terminal half of Kar1p. The potential Cdc31p-binding sequence of Kar1p contains three acidic amino acids which are not found in calmodulin-binding peptides, explaining the different substrate specificities of Cdc31p and calmodulin. Cdc31p was also able to bind to the carboxy terminus of Nuflp/Spc110p, another component of the SPB (Kilmartin, J. V., S. L. Dyos, D. Kershaw, and J. T. Finch. 1993. J. Cell Biol. 123:1175-1184). The association of Kar1p with the spindle pole body was independent of Cdc31p. Cdc31p, on the other hand, was not associated with SPBs of kar1 cells

A tailor-made purification strategy for oncolytic measles viruses using membrane-based processes

Cancer patients can benefit from the Measles virus, since in the early 70s a relation between cancer remission and an infection with Measles was first mentioned (Bluming, Ziegler 1971). Further studies confirmed this oncolytic activity and therefore, the Measles virus became highly interesting for the application in cancer treatment. However, for the widespread application as a therapeutic agent several bottlenecks have to be overcome in context of quantity and quality. For one therapeutic dose of oncolytic Measles viruses (OMV) at least 1011 infectious particles are needed (one vaccination contains ~103 TCID50) (Russell et al. 2014). Besides that, the impurities, such as host cell proteins (HCP) and host cell DNA (hcDNA), must be reduced to appropriate limits set by regulatory authorities. The full recovery of OMV infectivity must also addressed. This underlines the need of a tailor-made downstream processing. After we established a high titer production process, achieving OMV titers of 1011 TCID50 mL-1 (Grein et al. 2017), we are now focused on the downstream processing of OMV. For this purpose we characterized the OMV regarding process parameters used in DSP, such as stability towards ionic strength, osmolality, agglomeration and shear stress. Based on this, a clarification step was conducted, followed by the further purification with tangential flow filtration (TFF). By using polyether sulfone flat sheet membranes in concentration mode, we were able to recover the infectious virus and lowered the impurities by ~70% for hcDNA and ~80% for protein content. In the next purification step, we applied a discontinuous diafiltration and could deplete the impurities by ~95% in total. These results showed that TFF is an appropriate tool for the purification and formulation of OMV. References Bluming, Avrum Z.; Ziegler, John L. (1971): Regression of Burkitt\u27s Lymphoma in association with Measles infection. In The Lancet 298 (7715), pp. 105–106. DOI: 10.1016/S0140-6736(71)92086-1. Grein, Tanja A.; Schwebel, Felix; Kress, Marco; Loewe, Daniel; Dieken, Hauke; Salzig, Denise et al. (2017): Screening different host cell lines for the dynamic production of measles virus. In Biotechnology progress. DOI: 10.1002/btpr.2432. Russell, Stephen J.; Federspiel, Mark J.; Peng, Kah-Whye; Tong, Caili; Dingli, David; Morice, William G. et al. (2014): Remission of disseminated cancer after systemic oncolytic virotherapy. In Mayo Clinic proceedings 89 (7), pp. 926–933. DOI: 10.1016/j.mayocp.2014.04.003

Ultrafast Optical-Pump Terahertz-Probe Spectroscopy of the Carrier Relaxation and Recombination Dynamics in Epitaxial Graphene

The ultrafast relaxation and recombination dynamics of photogenerated electrons and holes in epitaxial graphene are studied using optical-pump Terahertz-probe spectroscopy. The conductivity in graphene at Terahertz frequencies depends on the carrier concentration as well as the carrier distribution in energy. Time-resolved studies of the conductivity can therefore be used to probe the dynamics associated with carrier intraband relaxation and interband recombination. We report the electron-hole recombination times in epitaxial graphene for the first time. Our results show that carrier cooling occurs on sub-picosecond time scales and that interband recombination times are carrier density dependent.Comment: 4 pages, 5 figure

A Comparative Genomic Analysis of Energy Metabolism in Sulfate Reducing Bacteria and Archaea

The number of sequenced genomes of sulfate reducing organisms (SRO) has increased significantly in the recent years, providing an opportunity for a broader perspective into their energy metabolism. In this work we carried out a comparative survey of energy metabolism genes found in 25 available genomes of SRO. This analysis revealed a higher diversity of possible energy conserving pathways than classically considered to be present in these organisms, and permitted the identification of new proteins not known to be present in this group. The Deltaproteobacteria (and Thermodesulfovibrio yellowstonii) are characterized by a large number of cytochromes c and cytochrome c-associated membrane redox complexes, indicating that periplasmic electron transfer pathways are important in these bacteria. The Archaea and Clostridia groups contain practically no cytochromes c or associated membrane complexes. However, despite the absence of a periplasmic space, a few extracytoplasmic membrane redox proteins were detected in the Gram-positive bacteria. Several ion-translocating complexes were detected in SRO including H+-pyrophosphatases, complex I homologs, Rnf, and Ech/Coo hydrogenases. Furthermore, we found evidence that cytoplasmic electron bifurcating mechanisms, recently described for other anaerobes, are also likely to play an important role in energy metabolism of SRO. A number of cytoplasmic [NiFe] and [FeFe] hydrogenases, formate dehydrogenases, and heterodisulfide reductase-related proteins are likely candidates to be involved in energy coupling through electron bifurcation, from diverse electron donors such as H2, formate, pyruvate, NAD(P)H, β-oxidation, and others. In conclusion, this analysis indicates that energy metabolism of SRO is far more versatile than previously considered, and that both chemiosmotic and flavin-based electron bifurcating mechanisms provide alternative strategies for energy conservation

An isotopic effect in phi photoproduction at a few GeV

A distinct isotopic effect in phi photoproduction at 2-5 GeV region is identified by examining the production amplitudes due to Pomeron-exchange and meson-exchange mechanisms. This effect is mainly caused by the pi-eta interference constrained by SU(3) symmetry and the isotopic structure of the gamma NN coupling in the direct phi-radiation amplitude. It can be tested experimentally by measuring differences in the polarization observables between the gamma-p and gamma-n reactions.Comment: 11 pages, 6 figure

Rumors of disease in the global village: outbreak verification.

Emerging infectious diseases and the growth of information technology have produced new demands and possibilities for disease surveillance and response. Increasing numbers of outbreak reports must be assessed rapidly so that control efforts can be initiated and unsubstantiated reports can be identified to protect countries from unnecessary economic damage. The World Health Organization has set up a process for timely outbreak verification to convert large amounts of data into accurate information for suitable action. We describe the context and processes of outbreak verification and information dissemination

Large-area NbN superconducting nanowire avalanche photon detectors with saturated detection efficiency

Superconducting circuits comprising SNSPDs placed in parallel—superconducting nanowire avalanche photodetectors, or SNAPs—have previously been demonstrated to improve the output signal-to-noise ratio (SNR) by increasing the critical current. In this work, we employ a 2-SNAP superconducting circuit with narrow (40 nm) niobium nitride (NbN) nanowires to improve the system detection efficiency to near-IR photons while maintaining high SNR. Additionally, while previous 2-SNAP demonstrations have added external choke inductance to stabilize the avalanching photocurrent, we show that the external inductance can be entirely folded into the active area by cascading 2-SNAP devices in series to produce a greatly increased active area. We fabricated series-2-SNAP (s2-SNAP) circuits with a nanowire length of 20 μm with cascades of 2-SNAPs providing the choke inductance necessary for SNAP operation. We observed that (1) the detection efficiency saturated at high bias currents, and (2) the 40 nm 2-SNAP circuit critical current was approximately twice that for a 40 nm non-SNAP configuration.United States. Dept. of Defense. Assistant Secretary of Defense for Research & Engineering (United States. Air Force Contract FA8721-05-C-0002

Effects of impurity scattering on electron-phonon resonances in semiconductor superlattice high-field transport

A non-equilibrium Green's function method is applied to model high-field quantum transport and electron-phonon resonances in semiconductor superlattices. The field-dependent density of states for elastic (impurity) scattering is found non-perturbatively in an approach which can be applied to both high and low electric fields. I-V curves, and specifically electron-phonon resonances, are calculated by treating the inelastic (LO phonon) scattering perturbatively. Calculations show how strong impurity scattering suppresses the electron-phonon resonance peaks in I-V curves, and their detailed sensitivity to the size, strength and concentration of impurities.Comment: 7 figures, 1 tabl