Preferential Colonization of Metastases by Oncolytic Vaccinia Virus Strain GLV-1h68 in a Human PC-3 Prostate Cancer Model in Nude Mice

Recently, we showed that the oncolytic vaccinia virus GLV-1h68 has a significant therapeutic potential in treating lymph node metastases of human PC-3 prostate carcinoma in tumor xenografts. In this study, underlying mechanisms of the virus-mediated metastases reduction were analyzed. Immunohistochemistry demonstrated that virus-treatment resulted in a drastically decrease of blood and lymph vessels, representing essential routes for PC-3 cell migration, in both tumors and metastases. Thus, GLV-1h68 drastically reduced essential routes for the metastatic spread of PC-3 cells. Furthermore, analysis of viral distribution in GLV-1h68-injected tumor-bearing mice by plaque assays, revealed significantly higher virus titers in metastases compared to solid tumors. To elucidate conditions potentially mediating the preferential viral colonization and eradication of metastases, microenvironmental components of uninfected tumors and metastases were compared by microscopic studies. These analyses revealed that PC-3 lymph node metastases showed increased vascular permeability, higher proliferation status of tumor cells as determined by BrdU- and Ki-67 assays and lesser necrosis of PC-3 cells than solid tumors. Moreover, an increased number of immune cells (MHCII⁺/CD68⁺ macrophages, MHCII⁺/CD19⁺ B lymphocytes) combined with an up-regulated expression of pro-inflammatory cytokines was observed in metastases in comparison to primary PC-3 tumors. We propose that these microenvironmental components mediated the metastatic tropism of GLV-1h68. Therefore, vaccinia virus-based oncolytic virotherapy might offer a novel treatment of metastatic prostate carcinomas in humans.     

Real-time cross-correlation image analysis of early events in IgE receptor signaling

Signaling in mast cells and basophils is mediated through IgE and its high affinity cell surface receptor, FcepsilonRI. Crosslinking of the receptors by a cognate multivalent antigen leads to degranulation and release of mediators of the allergic immune response. Using multicolor fluorescence confocal microscopy, we probed the spatio-temporal dynamics of early events in the IgE receptor signal cascade. We monitored the recruitment of GFP-/CFP-labeled signaling proteins by acquiring sequential images with time resolution of 3 s during stimulation of RBL-2H3 mast cells with multivalent antigen. A fluorescent tag on the antigen allowed us to visualize the plasma membrane localization of crosslinked receptors, and fluorescent cholera toxin B served as a plasma membrane marker. We developed an automated image analysis scheme to quantify the recruitment of fluorescent intracellular proteins to the plasma membrane and to assess the time-dependent colocalization of these and other membrane-associated proteins with crosslinked receptors as measured by cross-correlation between the plasma membrane distributions of the two fluorophores. This automated method permits analysis of thousands of individual images from multiple experiments for each cross-correlation pair. We systematically applied this analysis to characterize stimulated interactions of IgE receptors with several signaling proteins, including the tyrosine kinases Lyn and Syk, and the adaptor protein LAT. Notably, for Syk-CFP we observed a rapid stimulated translocation to the plasma membrane but very little colocalization with aggregated receptors. Our results demonstrate the utility of this simple, automated method to monitor protein interactions quantitatively during cell signaling.     

Binding of the Heterogeneous Ribonucleoprotein K (hnRNP K) to the Epstein-Barr Virus Nuclear Antigen 2 (EBNA2) Enhances Viral LMP2A Expression

The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.     

The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex

Stabilization and processing of stalled replication forks is critical for cell survival and genomic integrity. We characterize a novel DNA repair heterodimer of Nse5 and Nse6, which are nonessential nuclear proteins critical for chromosome segregation in fission yeast. The Nse5/6 dimer facilitates DNA repair as part of the Smc5-Smc6 holocomplex (Smc5/6), the basic architecture of which we define. Nse5-Nse6 [corrected] (Nse5 and Nse6) [corrected] mutants display a high level of spontaneous DNA damage and mitotic catastrophe in the absence of the master checkpoint regulator Rad3 (hATR). Nse5/6 mutants are required for the response to genotoxic agents that block the progression of replication forks, acting in a pathway that allows the tolerance of irreparable UV lesions. Interestingly, the UV sensitivity of Nse5/6 [corrected] is suppressed by concomitant deletion of the homologous recombination repair factor, Rhp51 (Rad51). Further, the viability of Nse5/6 mutants depends on Mus81 and Rqh1, factors that resolve or prevent the formation of Holliday junctions. Consistently, the UV sensitivity of cells lacking Nse5/6 can be partially suppressed by overexpressing the bacterial resolvase RusA. We propose a role for Nse5/6 mutants in suppressing recombination that results in Holliday junction formation or in Holliday junction resolution.     

A comparison of reactive oxygen species metabolism in the rat aorta and vena cava: focus on xanthine oxidase

Reactive oxygen species (ROS) are important mediators in vascular biology. Venous function, although relevant to cardiovascular disease, is still understudied. We compared aspects of ROS metabolism between a major artery (the aorta) and a major vein (the vena cava, VC) of the rat, with the hypothesis that venous ROS metabolism would be overall increased compared with its arterial counterpart. Superoxide and hydrogen peroxide (H2O2) release in basal conditions was higher in VC compared with aorta. The antioxidant capacity for H2O2 was also higher in VC than in aorta. Exogenous superoxide induced a higher contraction in VC compared with aorta. Protein expression of three major ROS metabolizing enzymes, xanthine oxidase (XO), CuZn-SOD, and catalase, was higher in VC compared with aorta. Because XO seemed a likely source of the higher VC ROS levels, we examined it further and found higher mRNA expression and activity of XO in VC compared with aorta. We also investigated the impact of XO inhibition by allopurinol on aorta and VC functional responses to norepinephrine, ANG II, ET-1, and ACh. Maximal ET-1-mediated contraction was decreased by allopurinol in VC but not in the aorta. Our results suggest that there are overall differences in ROS metabolism between aorta and VC, with the latter operating normally at a higher set point, releasing but also being able to handle, higher ROS levels. We propose XO to be an important source for these differences. The result of this particular comparison may be reflective of a general arteriovenous contrast.     

The Root Extract of the Medicinal Plant Pelargonium sidoides Is a Potent HIV-1 Attachment Inhibitor

Global HIV-1 treatment would benefit greatly from safe herbal medicines with scientifically validated novel anti-HIV-1 activities. The root extract from the medicinal plant Pelargonium sidoides (PS) is licensed in Germany as the herbal medicine EPs®7630, with numerous clinical trials supporting its safety in humans. Here we provide evidence from multiple cell culture experiments that PS extract displays potent anti-HIV-1 activity. We show that PS extract protects peripheral blood mononuclear cells and macrophages from infection with various X4 and R5 tropic HIV-1 strains, including clinical isolates. Functional studies revealed that the extract from PS has a novel mode-of-action. It interferes directly with viral infectivity and blocks the attachment of HIV-1 particles to target cells, protecting them from virus entry. Analysis of the chemical footprint of anti-HIV activity indicates that HIV-1 inhibition is mediated by multiple polyphenolic compounds with low cytotoxicity and can be separated from other extract components with higher cytotoxicity. Based on our data and its excellent safety profile, we propose that PS extract represents a lead candidate for the development of a scientifically validated herbal medicine for anti-HIV-1 therapy with a mode-of-action different from and complementary to current single-molecule drugs.     

Wind Farm Facilities in Germany Kill Noctule Bats from Near and Far

Over recent years, it became widely accepted that alternative, renewable energy may come at some risk for wildlife, for example, when wind turbines cause large numbers of bat fatalities. To better assess likely populations effects of wind turbine related wildlife fatalities, we studied the geographical origin of the most common bat species found dead below German wind turbines, the noctule bat (Nyctalus noctula). We measured stable isotope ratios of non-exchangeable hydrogen in fur keratin to separate migrants from local individuals, used a linear mixed-effects model to identify temporal, spatial and biological factors explaining the variance in measured stable isotope ratios and determined the geographical breeding provenance of killed migrants using isoscape origin models. We found that 72% of noctule bat casualties (n = 136) were of local origin, while 28% were long-distance migrants. These findings highlight that bat fatalities at German wind turbines may affect both local and distant populations. Our results indicated a sex and age-specific vulnerability of bats towards lethal accidents at turbines, i.e. a relatively high proportion of killed females were recorded among migratory individuals, whereas more juveniles than adults were recorded among killed bats of local origin. Migratory noctule bats were found to originate from distant populations in the Northeastern parts of Europe. The large catchment areas of German wind turbines and high vulnerability of female and juvenile noctule bats call for immediate action to reduce the negative cross-boundary effects of bat fatalities at wind turbines on local and distant populations. Further, our study highlights the importance of implementing effective mitigation measures and developing species and scale-specific conservation approaches on both national and international levels to protect source populations of bats. The efficacy of local compensatory measures appears doubtful, at least for migrant noctule bats, considering the large geographical catchment areas of German wind turbines for this species.     

Targeting conserved water molecules: Design of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine Hsp90 inhibitors using fragment-based screening and structure-based optimization

Inhibitors of the Hsp90 molecular chaperone are showing promise as anti-cancer agents. Here we describe a series of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors that were identified following structure-driven optimization of purine hits revealed by NMR based screening of a proprietary fragment library. Ligand-Hsp90 X-ray structures combined with molecular modeling led to the rational displacement of a conserved water molecule leading to enhanced affinity for Hsp90 as measured by fluorescence polarization, isothermal titration calorimetry and surface plasmon resonance assays. This displacement was achieved with a nitrile group, presenting an example of efficient gain in binding affinity with minimal increase in molecular weight. Some compounds in this chemical series inhibit the proliferation of human cancer cell lines in vitro and cause depletion of oncogenic Hsp90 client proteins and concomitant elevation of the co-chaperone Hsp70. In addition, one compound was demonstrated to be orally bioavailable in the mouse. This work demonstrates the power of structure-based design for the rapid evolution of potent Hsp90 inhibitors and the importance of considering conserved water molecules in drug design.     

Physiological homogeneity among the endosymbionts of Riftia pachyptila and Tevnia jerichonana revealed by proteogenomics

The two closely related deep-sea tubeworms Riftia pachyptila and Tevnia jerichonana both rely exclusively on a single species of sulfide-oxidizing endosymbiotic bacteria for their nutrition. They do, however, thrive in markedly different geochemical conditions. A detailed proteogenomic comparison of the endosymbionts coupled with an in situ characterization of the geochemical environment was performed to investigate their roles and expression profiles in the two respective hosts. The metagenomes indicated that the endosymbionts are genotypically highly homogeneous. Gene sequences coding for enzymes of selected key metabolic functions were found to be 99.9% identical. On the proteomic level, the symbionts showed very consistent metabolic profiles, despite distinctly different geochemical conditions at the plume level of the respective hosts. Only a few minor variations were observed in the expression of symbiont enzymes involved in sulfur metabolism, carbon fixation and in the response to oxidative stress. Although these changes correspond to the prevailing environmental situation experienced by each host, our data strongly suggest that the two tubeworm species are able to effectively attenuate differences in habitat conditions, and thus to provide their symbionts with similar micro-environments.