Rapid selection of specific MAP kinase-binders from designed ankyrin repeat protein libraries

We describe here the rapid selection of specific MAP-kinase binders from a combinatorial library of designed ankyrin repeat proteins (DARPins). A combined in vitro/in vivo selection approach, based on ribosome display and the protein fragment complementation assay (PCA), yielded a large number of different binders that are fully functional in the cellular cytoplasm. Ribosome-display selection pools of four successive selection rounds were examined to monitor the enrichment of JNK2-specific DARPins. Surprisingly, only one round of ribosome display with subsequent PCA selection of this pool was necessary to isolate a first specific binder with micromolar affinity. After only two rounds of ribosome-display selection followed by PCA, virtually all DARPins showed JNK2-specific binding, with affinities in the low nanomolar range. The enrichment factor of ribosome display thus approaches 105 per round. In a second set of experiments, similar results were obtained with the kinases JNK1 and p38 as targets. Again, almost all investigated DARPins obtained after two rounds of ribosome display showed specific binding to the targets used, JNK1 or p38. In all three selection experiments the identified DARPins possess very high specificity for the target kinase. Taken together, the combination of ribosome display and PCA selections allowed the identification of large pools of binders at unparalleled speed. Furthermore, DARPins are applicable in intracellular selections and immunoprecipitations from the extract of eukaryotic cell

The antibody-mediated targeted delivery of interleukin-10 inhibits endometriosis in a syngeneic mouse model

BACKGROUND Endometriosis is still a highly underdiagnosed disease, and the current medical and surgical treatment of endometriosis is associated with a high recurrence rate. This study investigates the use of derivatives of the human antibody F8, specific to the alternatively spliced extra-domain A of fibronectin (Fn), for the imaging and treatment of endometriosis. METHODS Immunohistochemistry and immunofluorescence was used to evaluate antigen expression in endometriotic tissue of human endometriosis and of a syngeneic mouse model of the disease. The in vivo targeting performance of a fluorescent derivative of the F8 antibody was assessed by imaging mice with endometriosis using a near-infrared fluorescence imager, 24 h following i.v. injection of the antibody conjugate. Furthermore, the mouse model was used for therapy experiments using two recombinant F8-based immunocytokines [F8-interleukin-10 (IL10) and F8-IL2] or saline for the treatment groups. RESULTS A very strong vascular expression of splice isoforms of Fn and of tenascin-C was observed in human endometriotic lesions by immunohistochemistry and immunofluorescence techniques. After i.v. administration, a selective accumulation of the F8 antibody in endometriotic lesions could be observed in a syngeneic mouse model. These targeting data were used as a basis for therapy experiments with a pro-inflammatory (F8-IL2) and an anti-inflammatory (F8-IL10) cytokine fusion protein of the F8 antibody. The average lesion size in the F8-IL10 treatment group was clearly reduced compared with the saline control group and with the F8-IL2 group, for which no therapeutic effects were observed. CONCLUSIONS The F8 antibody targets endometriotic lesions in vivo in a mouse model of endometriosis and may be used for the non-invasive imaging of the disease and for the pharmacodelivery of anti-inflammatory cytokines, such as IL1

Doppler-free high resolution continuous wave optical UV-spectroscopy on the \mathrm{A}\,^2\Sigma^+ \leftarrow \mathrm{X}\,^2\Pi_{3/2} transition in nitric oxide

We report on Doppler-free continuous-wave optical UV-spectroscopy resolving the hyperfine structure of the \mathrm{A}\,^2\Sigma^+ \leftarrow \mathrm{X}\,^2\Pi_{3/2} transition in nitric oxide for total angular momenta $J_X=1.5-19.5$ on the $\mathrm{oP_{12ee}}$ branch. The resulting line splittings are compared to calculated splittings and fitted determining new values for the molecular constants $b, c, eQq_0$ and $b_F$ for the \mathrm{A}\,^2\Sigma^+ state. The constants are in good agreement with values previously determined by quantum beat spectroscopy.Comment: 8 Pages, 4 figure

Diurnal cycle of linear contrails, cirrus, and outgoing longwave radiation in the North Atlantic from MODIS, MSG and models

Cloud cover from linear contrails, cirrus cloud cover, and outgoing longwave radiation (OLR) at top of the atmosphere were derived from MSG SEVIRI in a North Atlantic region (NAR) for a period of eight years (Febr 2004- Jan 2012) with 15 min time resolution. The aviation induced contributions to cirrus coverage and OLR in the NAR flight corridor were derived from these data assuming linear response of cirrus and OLR changes to air traffic density and cirrus/OLR background without aviation assumed either constant of as observed in the corresponding South Atlantic region (SAR). Global results were obtained by extrapolating the regional results with global models [Graf et al., 2012; Schumann and Graf, 2013]. Here, the results are compared with linear contrail coverage values derived from MODIS aboard the LEO satellites Terra and Aqua [Duda, Minnis et al., 2013] . The global observations revealed surprisingly high contrail cover in the North Atlantic region. Terra and Aqua overpass times in the NAR are limited to four narrow time intervals and hence cannot resolve the full diurnal cycle. The results are also compared with predictions of contrail cover and OLR forecast by the Contrail and Cirrus Prediction tool CoCiP. CoCiP computes the cirrus and OLR changes for given meteorology and given air traffic. The comparisons show that the LEO observations tend to overestimate the daily mean aviation effects in the NAR because the observations times coincide with times of traffic peaks in the NAR

Increasing variability of body mass and health correlates in Swiss conscripts, a possible role of relaxed natural selection?

Background and objectives The body mass index (BMI) is an established anthropometric index for the development of obesity related conditions. However, little is known about the distribution of BMI within a population, especially about this distribution’s temporal change. Here, we analysed changes in the distribution of height, weight and BMI over the past 140 years based on data of Swiss conscripts and tested for correlations between anthropometric data and standard blood parameters. Methods Height and weight were measured in 59,504 young Swiss males aged 18-19 years during conscription in 1875-79, 1932-36, 1994 and 2010-12. For 65% of conscripts in 2010-12 results of standard blood analysis were available. We calculated descriptive statistics of the distribution of height, weight, and BMI over the four time periods and tested for associations between BMI and metabolic parameters. Results Average and median body height, body weight and BMI increased over time. Height did no longer increase between 1994 and 2010-12, while weight and BMI still increased over these two decades. Variability ranges of weight and BMI increased over time, while variation of body height remained constant. Elevated levels of metabolic and inflammatory blood parameters were found at both ends of BMI distribution. Conclusions and Implications Both overweight and underweight subgroups showed similar changes in inflammation parameters, pointing towards related metabolic deficiencies in both conditions. In addition to environmental influences, our results indicate a potential role of relaxed natural selection on genes affecting metabolism and body composition

Collisional shift and broadening of Rydberg states in nitric oxide at room temperature

We report on the collisional shift and line broadening of Rydberg states in nitric oxide (NO) with increasing density of a background gas at room temperature. As a background gas we either use NO itself or nitrogen (N$_{2}$). The precision spectroscopy is performed by a sub-Doppler three-photon excitation scheme with a subsequent readout of the Rydberg states realized by the amplification of a current generated by free charges due to collisions. The shift shows a dependence on the rotational quantum state of the ionic core and no dependence on the principle quantum number of the orbiting Rydberg electron. The experiment was performed in the context of developing a trace-gas sensor for breath-gas analysis in a medical application