Heterologous production of five Hepatitis C virus-derived antigens in three Saccharomyces cerevisiae host strains

In this study, the production of recombinant Hepatitis C virus (HCV) derived proteins from transformed Saccharomyces cerevisiae yeast cells is reported. Three different yeast strains (GRF18U, BY4743-4A and CENPK 113-5D) have been transformed for the intracellular expression of five antigens of different dimensions (from 32.8 to 85.2 kDa), all derived from the non-structural (NS) region of different HCV viruses' genotypes and posed under the control of a glycolytic promoter. The putative trans-membrane domains contained in four antigens seem responsible of their accumulation as protein aggregates. Good productions of the smaller and of the bigger antigens (50 and 30 mgl(-1), respectively) have been observed in simple flask batch cultures. Productions are strongly dependent from the genetic background of the yeast host and from the cellular localization of the antigen, while they appear independent from the growth rate of the transformed hosts. For every recombinant antigen tested, the highest production levels were achieved with the CENPK 113-5D-host strain, while the GRF18U strain shows symptoms of a heavily stressed phenotype.     

High Energy Digestion Efficiency and Altered Lipid Metabolism Contribute to Obesity in BFMI Mice

To constitute a valuable resource to identify individual genes involved in the development of obesity, a novel mouse model, the Berlin Fat Mouse Inbred line 860 (BFMI860), was established. In order to characterize energy intake and energy expenditure in obese BFMI860 mice, we performed two independent sets of experiments in male BFMI860 and B6 control mice (10 per line). In experiment 1, we analyzed body fat content noninvasively by dual‐energy X‐ray absorptiometry and measured resting metabolic rate at thermoneutrality (RMRt) and respiratory quotient (RQ) in week 6, 10, and 18. In a second experiment, energy digested (energy intake minus fecal energy loss) was determined by bomb calorimetry from week 6 through week 12. BFMI860 mice were heavier and had higher fat mass (final body fat content was 24.7% compared with 14.6% in B6). They also showed fatty liver syndrome. High body fat accumulation in BFMI860 mice was restricted to weeks 6–10 and was accompanied by hyperphagia, higher energy digestion, higher RQs, and abnormally high blood triglyceride levels. Lean mass–adjusted RMRt was not altered between lines. These results indicate that in BFMI860 mice, the excessive accumulation of body fat is associated with altered lipid metabolism, high energy intake, and energy digestion. Assuming that BFMI860 mice and their obese phenotypes are of polygenic nature, this line is an excellent model for the study of obesity in humans, especially for juvenile obesity and hyperlipidemia.     

Superparamagnetic iron oxide nanoparticles as radiosensitizer via enhanced reactive oxygen species formation

Internalization of citrate-coated and uncoated superparamagnetic iron oxide nanoparticles by human breast cancer (MCF-7) cells was verified by transmission electron microscopy imaging. Cytotoxicity studies employing metabolic and trypan blue assays manifested their excellent biocompatibility. The production of reactive oxygen species in iron oxide nanoparticle loaded MCF-7 cells was explained to originate from both, the release of iron ions and their catalytically active surfaces. Both initiate the Fenton and Haber-Weiss reaction. Additional oxidative stress caused by X-ray irradiation of MCF-7 cells was attributed to the increase of catalytically active iron oxide nanoparticle surfaces.     

Postnatal handling reverses social anxiety in serotonin receptor 1A knockout mice

Mice lacking the serotonin receptor 1A (Htr1a knockout, Htr1a ^KO ) show increased innate and conditioned anxiety. This phenotype depends on functional receptor activity during the third through fifth weeks of life and thus appears to be the result of long-term changes in brain function as a consequence of an early deficit in serotonin signaling. To evaluate whether this phenotype can be influenced by early environmental factors, we subjected Htr1a knockout mice to postnatal handling, a procedure known to reduce anxiety-like behavior and stress responses in adulthood. Offspring of heterozygous Htr1a knockout mice were separated from their mother and exposed 15 min each day from postnatal day 1 (PD1) to PD14 to clean bedding. Control animals were left undisturbed. Maternal behavior was observed during the first 13 days of life. Adult male offspring were tested in the open field, social approach and resident–intruder tests and assessed for corticosterone response to restraint stress. Knockout mice showed increased anxiety in the open field and in the social approach test as well as an enhanced corticosterone response to stress. However, while no effect of postnatal handling was seen in wild-type mice, handling reduced anxiety-like behavior in the social interaction test and the corticosterone response to stress in knockout mice. These findings extend the anxiety phenotype of Htr1a ^KO mice to include social anxiety and demonstrate that this phenotype can be moderated by early environmental factors.     

The EMBL Nucleotide Sequence Database: major new developments

The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/) incorporates, organizes and distributes nucleotide sequences from all available public sources. The database is located and maintained at the European Bioinformatics Institute (EBI) near Cambridge, UK. In an international collaboration with DDBJ (Japan) and GenBank (USA), data are exchanged amongst the collaborating databases on a daily basis to achieve optimal synchronization. Webin is the preferred web-based submission system for individual submitters, while automatic procedures allow incorporation of sequence data from large-scale genome sequencing centres and from the European Patent Office (EPO). Database releases are produced quarterly. Network services allow free access to the most up-to-date data collection via FTP, Email and World Wide Web interfaces. EBI's Sequence Retrieval System (SRS) integrates and links the main nucleotide and protein databases plus many other specialized molecular biology databases. For sequence similarity searching, a variety of tools (e.g. Fasta, BLAST) are available which allow external users to compare their own sequences against the latest data in the EMBL Nucleotide Sequence Database and SWISS-PROT. All resources can be accessed via the EBI home page at http://www.ebi.ac.uk.     

Which functional groups of the molybdopterin ligand should be considered when modeling the active sites of the molybdenum and tungsten cofactors? A density functional theory study

A density functional theory study of the influence of the various functional groups of the molybdopterin ligand on electronic and geometric properties of active-site models for the molybdenum and tungsten cofactors has been undertaken. We used analogous molybdenum and tungsten complexes with increasingly accurate representation of the molybdopterin ligands and compared bond lengths, angles, charge distribution, composition of the binding orbitals, as well as the redox potentials in relation to each other. On the basis of our findings, we suggest using ligand systems including the pyrane and the pyrazine rings, besides the dithiolene function, to obtain sufficiently reliable computational, but also synthetic, models for the molybdenum and tungsten cofactors, whereas the second ring of the pterin might be neglected for efficiency reasons.     

Quiescent and Active Hippocampal Neural Stem Cells with Distinct Morphologies Respond Selectively to Physiological and Pathological Stimuli and Aging

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Highlights? Notch signaling defines morphologically distinct hippocampal stem cell populations ? Radial and horizontal hippocampal stem cells depend on canonical Notch signaling ? Different neural stem cell subpopulations respond selectively to neurogenic stimuli ? Aging results in a reversible transition of stem cells to a quiescent state     

Development of tetravalent IgG1 dual targeting IGF-1R-EGFR antibodies with potent tumor inhibition

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.