Temperature-induced sign change of the magnetic interlayer coupling in Ni/Ni25Mn75/Ni trilayers on Cu3Au(001)

We investigated the magnetic interlayer coupling between two ferromagnetic (FM) Ni layers through an antiferromagnetic (AFM) Ni 25Mn75 layer and the influence of this coupling on the exchange bias phenomenon. The interlayer coupling energy of an epitaxial trilayer of 14 atomic monolayers (ML) Ni/45 ML Ni 25Mn75/16 ML Ni on Cu3Au(001) was extracted from minor-loop magnetization measurements using in-situ magneto-optical Kerr effect. The interlayer coupling changes from ferromagnetic to antiferromagnetic when the temperature is increased above 300 K. This sign change is interpreted as the result of the competition between an antiparallel Ruderman-Kittel-Kasuya-Yosida (RKKY)-type interlayer coupling, which dominates at high temperature, and a stronger direct exchange coupling across the AFM layer, which is present only below the Néel temperature of the AFM layer

Correlation dynamics between electrons and ions in the fragmentation of D2_2 molecules by short laser pulses

We studied the recollision dynamics between the electrons and D$_2^+$ ions following the tunneling ionization of D$_2$ molecules in an intense short pulse laser field. The returning electron collisionally excites the D$_2^+$ ion to excited electronic states from there D$_2^+$ can dissociate or be further ionized by the laser field, resulting in D$^+$ + D or D$^+$ + D$^+$, respectively. We modeled the fragmentation dynamics and calculated the resulting kinetic energy spectrum of D$^+$ to compare with recent experiments. Since the recollision time is locked to the tunneling ionization time which occurs only within fraction of an optical cycle, the peaks in the D$^+$ kinetic energy spectra provides a measure of the time when the recollision occurs. This collision dynamics forms the basis of the molecular clock where the clock can be read with attosecond precision, as first proposed by Corkum and coworkers. By analyzing each of the elementary processes leading to the fragmentation quantitatively, we identified how the molecular clock is to be read from the measured kinetic energy spectra of D$^+$ and what laser parameters be used in order to measure the clock more accurately.Comment: 13 pages with 14 figure

Intensification of MVA and influenza virus production through high-cell-density cultivation approaches

Background. Unlike production of recombinant proteins, continuous production of viral vaccines at high cell densities (HCD) is often constrained by a decrease in cell-specific virus yields, early host cell lysis during virus propagation and limited virus recovery from culture broth. Nevertheless, advanced fed-batch [1] and perfusion strategies can be applied to achieve high-yield virus production processes. In this study, the development of a semi-continuous process for the production of the modified vaccinia Ankara virus isolate MVA-CR19 and influenza virus A/PR/8/34 (H1N1) in HCD cultivations of the suspension cell line AGE1.CR.pIX (ProBioGen AG, Berlin) is presented. Methods. Depending on the required scale, high cell concentrations (~ 50×106 cells/mL) were achieved either through medium renewal by periodic centrifugation (semi-perfusion) in 50 mL cultivations or using an alternating tangential flow (ATF) perfusion system for 1 L bioreactors. Process development and optimization comprised three phases: 1) assessment of different fed-batch and medium exchange strategies for the propagation of MVA-CR19 or influenza A/PR/8/34 viruses in 50 mL cultivations; 2) scale-up and process optimization of the selected high-yield process strategy to a 1 L bioreactor with the ATF system, and 3) integration of a one-step purification process using magnetic sulfated cellulose particles (MSCP). For both viruses, conventional batch cultivation (no addition/medium exchange after infection) was compared with processes applying fed-batch, periodic medium exchange and the combination of both during virus propagation. Results. Perfusion and semi-perfusion at a feeding rate of 0.05 nL/cell×d was suitable to propagate AGE1.CR.pIX cells above 60×106 cells/mL with neither limitation nor overload of nutrients. For infections at 50 mL scale, the application of a combined strategy comprising an initial fed-batch phase followed by a periodic virus harvest phase resulted in the highest product yield with a more than 10-fold increase in virus particles concentration compared to the conventional batch processes operated at 4 to 8×106 cells/mL [2]. Additionally, a 3-fold increase in both cell-specific yield (virus particles/cell) and volumetric productivity (virus particles/L×d) could be obtained. Comparable yields were observed when up-scaling to a 1 L bioreactor using an ATF-system, even when virus particles were retained within the bioreactor. Further selection of the optimal pore size of the ATF membrane allowed semi-continuous harvesting of the produced viruses and its purification with MSCPs with a recovery from 30 to 50%. In all cases, cell-specific yields and volumetric productivities reached their maxima at 72 h post-infection, indicating that the process should be stopped at that time point. Conclusion. Compared to conventional batch processes, the developed HCD process offers significantly higher productivities including the option to integrate a one-step purification process in a semi-continuous mode. Overall, the results show that there is a great potential for semi-continuous HCD processes for the production of viral vaccines in larger scales, which could support efforts towards the establishment of continuous vaccine manufacturing. References. 1. Pohlscheidt, M., et al., Development and optimisation of a procedure for the production of Parapoxvirus ovis by large-scale microcarrier cell culture in a non-animal, non-human and non-plant-derived medium. Vaccine, 2008. 26(12): p. 1552-65. 2. Lohr, V., et al., New avian suspension cell lines provide production of influenza virus and MVA in serum-free media: studies on growth, metabolism and virus propagation. Vaccine, 2009. 27(36): p. 4975-82

Process optimization for semi-continuous virus production at high cell densities

Background. Unlike production of recombinant proteins, continuous production of viral vaccines at high cell densities (HCD) is still constrained by host cell lysis during virus propagation and limited virus recovery from culture broth. Nevertheless, advanced fed-batch [1] and perfusion strategies can be applied to achieve a high-yield virus production processes. In this study, the development of a high-yield semi-continuous process for the production and purification of the modified vaccinia Ankara virus isolate MVA-CR19 and influenza A/PR/8 in HCD cultivations of the suspension cell line AGE1.CR.pIX (ProBioGen AG, Berlin) is presented. Methods. Depending on the required scale, high cell concentrations (~ 50×106 cell mL-1) were achieved either through medium renewal by periodic centrifugation (semi-perfusion) in 50 mL cultivations or using an alternating tangential flow (ATF) perfusion system for 1 L bioreactors. Process development and optimization comprised three phases: 1) assessment of different fed-batch and medium exchange strategies for the propagation of MVA-CR19 or influenza A/PR/8 viruses in 50 mL cultivations; 2) scale-up and process optimization of the high-yield process strategy to a 1 L bioreactor with the ATF system, and 3) integration of a purification process step using magnetic sulfated cellulose particles (MSCP). For both viruses, conventional batch cultivation (no addition/medium exchange after infection) was compared with processes applying fed-batch, periodic medium exchange and the combination of both during virus propagation. Results. Perfusion and semi-perfusion at a feeding rate of 0.05 nL/cell×d was suitable to propagate AGE1.CR.pIX cells above 60×106 cells/mL with neither limitation nor overload of nutrients. For infections in 50 mL, the application of a combined strategy comprising an initial fed-batch phase followed by a periodic virus harvest phase resulted in the highest product yield with a more than 10-fold increase, compared to the conventional batch processes at 4 to 8×106 cell/mL [2]. Additionally, a 3-fold increase in both cell-specific yield (virus/cell) and volumetric productivity (virus/L×d) could be obtained. Although product harvesting was suboptimal when up-scaling to a 1 L bioreactor with ATF-system, comparable increases in virus yields and productivity with respect to the conventional batch process were observed. In all cases, cell-specific yields and volumetric productivities reached their peak values at the peak virus concentrations, indicating that the process should be stopped at that time point. Eventually, selection of the optimal pore size of the membrane of the ATF-system allowed semi-continuous harvesting of the produced viruses and its purification with MSCPs with a recovery of about 50%. Conclusion. Compared to conventional batch processes, the developed HCD process offers significantly higher productivities including the option to integrate a purification step in a semi-continuous mode. Overall, the results show that there is a great potential for semi-continuous HCD processes for the production of viral vaccines in larger scales, which could intensify the discussion towards the establishment of true continuous production process

Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles

Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD) human (HAd) and canine (CAV-2) adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV) effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS). With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways - but in opposite directions - suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer

Connexin 43 mediated gap junctional communication enhances breast tumor cell diapedesis in culture

INTRODUCTION: Metastasis involves the emigration of tumor cells through the vascular endothelium, a process also known as diapedesis. The molecular mechanisms regulating tumor cell diapedesis are poorly understood, but may involve heterocellular gap junctional intercellular communication (GJIC) between tumor cells and endothelial cells. METHOD: To test this hypothesis we expressed connexin 43 (Cx43) in GJIC-deficient mammary epithelial tumor cells (HBL100) and examined their ability to form gap junctions, establish heterocellular GJIC and migrate through monolayers of human microvascular endothelial cells (HMVEC) grown on matrigel-coated coverslips. RESULTS: HBL100 cells expressing Cx43 formed functional heterocellular gap junctions with HMVEC monolayers within 30 minutes. In addition, immunocytochemistry revealed Cx43 localized to contact sites between Cx43 expressing tumor cells and endothelial cells. Quantitative analysis of diapedesis revealed a two-fold increase in diapedesis of Cx43 expressing cells compared to empty vector control cells. The expression of a functionally inactive Cx43 chimeric protein in HBL100 cells failed to increase migration efficiency, suggesting that the observed up-regulation of diapedesis in Cx43 expressing cells required heterocellular GJIC. This finding is further supported by the observation that blocking homocellular and heterocellular GJIC with carbenoxolone in co-cultures also reduced diapedesis of Cx43 expressing HBL100 tumor cells. CONCLUSION: Collectively, our results suggest that heterocellular GJIC between breast tumor cells and endothelial cells may be an important regulatory step during metastasis

Comparison of Recombinant Human Haptocorrin Expressed in Human Embryonic Kidney Cells and Native Haptocorrin

Haptocorrin (HC) is a circulating corrinoid binding protein with unclear function. In contrast to transcobalamin, the other transport protein in blood, HC is heavily glycosylated and binds a variety of cobalamin (Cbl) analogues. HC is present not only in blood but also in various secretions like milk, tears and saliva. No recombinant form of HC has been described so far. We report the expression of recombinant human HC (rhHC) in human embryonic kidney cells. We purified the protein with a yield of 6 mg (90 nmol) per litre of cell culture supernatant. The isolated rhHC behaved as native HC concerning its spectral properties and ability to recognize both Cbl and its baseless analogue cobinamide. Similar to native HC isolated from blood, rhHC bound to the asialoglycoprotein receptor only after removal of terminal sialic acid residues by treatment with neuraminidase. Interestingly, rhHC, that compared to native HC contains four excessive amino acids (…LVPR) at the C-terminus, showed subtle changes in the binding kinetics of Cbl, cobinamide and the fluorescent Cbl conjugate CBC. The recombinant protein has properties very similar to native HC and although showing slightly different ligand binding kinetics, rhHC is valuable for further biochemical and structural studies