Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound

AMP397 is a novel antiepileptic agent and the first competitive AMPA antagonist with high receptor affinity, good in vivo potency, and oral activity. AMP397 has a structural alert (aromatic nitro group) and was mutagenic in Salmonella typhimurium strains TA97a, TA98 and TA100 without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. The amino derivative of AMP397 was negative in wild-type strains TA98 and TA100. AMP397 was negative in a mouse lymphoma tk assay, which included a 24h treatment without S9. A weak micronucleus induction in vitro was found at the highest concentrations tested in V79 cells with S9. AMP397 was negative in the following in vivo studies, which included the maximum tolerated doses of 320mg/kg in mice and 2000mg/kg in rats: MutaMouse assay in colon and liver (5x320mg/kg) at three sampling times (3, 7 and 31 days after the last administration); DNA binding study in the liver of mice and rats after a single treatment with [14C]-AMP397; comet assay (1x2000mg/kg) in jejunum and liver of rats, sampling times 3 and 24h after administration; micronucleus test (2x320mg/kg) in the bone marrow of mice, sampling 24h after the second administration. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. In particular, no genotoxic metabolite is formed in mammalian cells, and, if formed by intestinal bacteria, is unable to exert any genotoxic activity in the adjacent intestinal tissue. These data were considered to provide sufficient safety to initiate clinical development of the compound.     

An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo

pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.     

Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework

Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells. These so-called intrabodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or by blocking interactions with other proteins. However, the intracellular expression and activity of many single-chain Fvs are limited by their instability and folding efficiency in the reducing intracellular environment, where the highly conserved intrachain disulfide bonds do not form. In the present work, we used an in vivo selection system to isolate novel antigen-binding intrabodies. We screened two intrabody libraries carrying a randomized third hypervariable loop onto the heavy chain of a stable framework, which had been further optimized by random mutagenesis for better behavior in the selection system, and we biophysically characterized the selected variants to interpret the outcome of the selection. Our results show that single-framework intrabody libraries can be directly screened in vivo to rapidly select antigen-specific intrabodies.     

Homothorax switches function of Drosophila photoreceptors from color to polarized light sensors

Different classes of photoreceptors (PRs) allow animals to perceive various types of visual information. In the Drosophila eye, the outer PRs of each ommatidium are involved in motion detection while the inner PRs mediate color vision. In addition, flies use a specialized class of inner PRs in the "dorsal rim area" of the eye (DRA) to detect the e-vector of polarized light, allowing them to exploit skylight polarization for orientation. We show that homothorax is both necessary and sufficient for inner PRs to adopt the polarization-sensitive DRA fate instead of the color-sensitive default state. Homothorax increases rhabdomere size and uncouples R7-R8 communication to allow both cells to express the same opsin rather than different ones as required for color vision. Homothorax expression is induced by the iroquois complex and the wingless (wg) pathway. However, crucial wg pathway components are not required, suggesting that additional signals are involved.     

Guanine-Modified Inhibitory Oligonucleotides Efficiently Impair TLR7- and TLR9-Mediated Immune Responses of Human Immune Cells

Activation of TLR7 and TLR9 by endogenous RNA- or DNA-containing ligands, respectively, is thought to contribute to the complicated pathophysiology of systemic lupus erythematosus (SLE). These ligands induce the release of type-I interferons by plasmacytoid dendritic cells and autoreactive antibodies by B-cells, both responses being key events in perpetuating SLE. We recently described the development of inhibitory oligonucleotides (INH-ODN), which are characterized by a phosphorothioate backbone, a CC(T)XXX_3–5GGG motif and a chemical modification of the G-quartet to avoid the formation of higher order structures via intermolecular G-tetrads. These INH-ODNs were equally or significantly more efficient to impair TLR7- and TLR9-stimulated murine B-cells, macrophages, conventional and plasmacytoid dendritic cells than the parent INH-ODN 2088, which lacks G-modification. Here, we evaluate the inhibitory/therapeutic potential of our set of G-modified INH-ODN on human immune cells. We report the novel finding that G-modified INH-ODNs efficiently inhibited the release of IFN-α by PBMC stimulated either with the TLR7-ligand oligoribonucleotide (ORN) 22075 or the TLR9-ligand CpG-ODN 2216. G-modification of INH-ODNs significantly improved inhibition of IL-6 release by PBMCs and purified human B-cells stimulated with the TLR7-ligand imiquimod or the TLR9-ligand CpG-ODN 2006. Furthermore, inhibition of B-cell activation analyzed by expression of activation markers and intracellular ATP content was significantly improved by G-modification. As observed with murine B-cells, high concentrations of INH-ODN 2088 but not of G-modified INH-ODNs stimulated IL-6 secretion by PBMCs in the absence of TLR-ligands thus limiting its blocking efficacy. In summary, G-modification of INH-ODNs improved their ability to impair TLR7- and TLR9-mediated signaling in those human immune cells which are considered as crucial in the pathophysiology of SLE.     

Pre-Use Susceptibility to Ceftaroline in Clinical Staphylococcus aureus Isolates from Germany: Is There a Non-Susceptible Pool to be Selected?

Ceftaroline is a new cephalosporin active against Methicillin-resistant Staphylococcus aureus (MRSA). Based on a representative collection of clinical S. aureus isolates from Germany, supplemented with isolates of clonal lineages ST228 and ST239, we demonstrate the in-vitro susceptibility towards ceftaroline prior to its introduction into clinical use for a total of 219 isolates. Susceptibility testing was performed by broth microdilution, disc diffusion and Etest, respectively. Results were interpreted according to EUCAST guidelines and showed considerable variance in dependence on clonal affiliation of the isolates tested. Among isolates of widespread hospital-associated lineages we found a high proportion of clinical isolates with MICs close to the EUCAST breakpoint (MIC_50/90 1.0/1.5 mg/L); currently, interpretation of these “borderline” MICs is complicated by a lack of concordant susceptibility testing methods and reasonable breakpoint determination. Isolates of clonal lineages ST228 and ST239 demonstrated increased MIC_50/90 values of 2.5/3.33 mg/L. Sequencing of mecA revealed no association of resistance to a specific mecA polymorphism, but rather reveals two regions in the non-penicillin-binding domain of PbP2a which displayed different combinations of mutations putatively involved in resistance development. This study provides national baseline data to (i) adjust susceptibility testing methods and current breakpoints to clinical and epidemiological requirements, (ii) evaluate current breakpoints with respect to therapeutic outcome and (iii) monitor further resistance evolution.     

Genotypic Tannin Levels in Populus tremula Impact the Way Nitrogen Enrichment Affects Growth and Allocation Responses for Some Traits and Not for Others

Plant intraspecific variability has been proposed as a key mechanism by which plants adapt to environmental change. In boreal forests where nitrogen availability is strongly limited, nitrogen addition happens indirectly through atmospheric N deposition and directly through industrial forest fertilization. These anthropogenic inputs of N have numerous environmental consequences, including shifts in plant species composition and reductions in plant species diversity. However, we know less about how genetic differences within plant populations determine how species respond to eutrophication in boreal forests. According to plant defense theories, nitrogen addition will cause plants to shift carbon allocation more towards growth and less to chemical defense, potentially enhancing vulnerability to antagonists. Aspens are keystone species in boreal forests that produce condensed tannins to serve as chemical defense. We conducted an experiment using ten Populus tremula genotypes from the Swedish Aspen Collection that express extreme levels of baseline investment into foliar condensed tannins. We investigated whether investment into growth and phenolic defense compounds in young plants varied in response to two nitrogen addition levels, corresponding to atmospheric N deposition and industrial forest fertilization. Nitrogen addition generally caused growth to increase, and tannin levels to decrease; however, individualistic responses among genotypes were found for height growth, biomass of specific tissues, root:shoot ratios, and tissue lignin and N concentrations. A genotype’s baseline ability to produce and store condensed tannins also influenced plant responses to N, although this effect was relatively minor. High-tannin genotypes tended to grow less biomass under low nitrogen levels and more at the highest fertilization level. Thus, the ability in aspen to produce foliar tannins is likely associated with a steeper reaction norm of growth responses, which suggests a higher plasticity to nitrogen addition, and potentially an advantage when adapting to higher concentrations of soil nitrogen.     

U.S. National Institutes of Health Core Consolidation–Investing in Greater Efficiency

The U.S. National Institutes of Health (NIH) invests substantial resources in core research facilities (cores) that support research by providing advanced technologies and scientific and technical expertise as a shared resource. In 2010, the NIH issued an initiative to consolidate multiple core facilities into a single, more efficient core. Twenty-six institutions were awarded supplements to consolidate a number of similar core facilities. Although this approach may not work for all core settings, this effort resulted in consolidated cores that were more efficient and of greater benefit to investigators. The improvements in core operations resulted in both increased services and more core users through installation of advanced instrumentation, access to higher levels of management expertise; integration of information management and data systems; and consolidation of billing; purchasing, scheduling, and tracking services. Cost recovery to support core operations also benefitted from the consolidation effort, in some cases severalfold. In conclusion, this program of core consolidation resulted in improvements in the effective operation of core facilities, benefiting both investigators and their supporting institutions.