Improved Elution Conditions for Native Co-Immunoprecipitation

Native immunoprecipitation followed by protein A-mediated recovery of the immuno-complex is a powerful tool to study protein-protein interactions. A limitation of this technique is the concomitant recovery of large amounts of immunoglobulin, which interferes with down-stream applications such as mass spectrometric analysis and Western blotting. Here we report a detergent-based "soft" elution protocol that allows effective recovery of immunoprecipitated antigen and binding partners, yet avoids elution of the bulk of the immunoglobulin.We assessed the performance of the soft elution protocol using immunoprecipitation of Adaptor protein complex 1 (AP-1) and associated proteins as a test case. Relative to conventional elution conditions, the novel protocol substantially improved the sensitivity of mass spectrometric identification of immunoprecipitated proteins from unfractionated solution digests. Averaging over three independent experiments, Mascot scores of identified AP-1 binding partners were increased by 39%. Conversely, the estimated amount of recovered immunoglobulin was reduced by 44%. We tested the protocol with five further antibodies derived from rabbit, mouse and goat. In each case we observed a significant reduction of co-eluting immunoglobulin.The soft elution protocol presented here shows superior performance compared to standard elution conditions for subsequent protein identification by mass spectrometry from solution digests. The method was developed for rabbit polyclonal antibodies, but also performed well with the tested goat and mouse antibodies. Hence we expect the soft elution protocol to be widely applicable

The Pairwise Peculiar Velocity Dispersion of Galaxies: Effects of the Infall

We study the reliability of the reconstruction method which uses a modelling of the redshift distortions of the two-point correlation function to estimate the pairwise peculiar velocity dispersion of galaxies. In particular, the dependence of this quantity on different models for the infall velocity is examined for the Las Campanas Redshift Survey. We make extensive use of numerical simulations and of mock catalogs derived from them to discuss the effect of a self-similar infall model, of zero infall, and of the real infall taken from the simulation. The implications for two recent discrepant determinations of the pairwise velocity dispersion for this survey are discussed.Comment: minor changes in the discussion; accepted for publication in ApJ; 8 pages with 2 figures include

The cross-correlation between galaxies of different luminosities and Colors

We study the cross-correlation between galaxies of different luminosities and colors, using a sample selected from the SDSS Dr 4. Galaxies are divided into 6 samples according to luminosity, and each of these samples is divided into red and blue subsamples. Projected auto-correlation and cross-correlation is estimated for these subsample. At projected separations r_p > 1\mpch, all correlation functions are roughly parallel, although the correlation amplitude depends systematically on luminosity and color. On r_p < 1\mpch, the auto- and cross-correlation functions of red galaxies are significantly enhanced relative to the corresponding power laws obtained on larger scales. Such enhancement is absent for blue galaxies and in the cross-correlation between red and blue galaxies. We esimate the relative bias factor on scales r > 1\mpch for each subsample using its auto-correlation function and cross-correlation functions. The relative bias factors obtained from different methods are similar. For blue galaxies the luminosity-dependence of the relative bias is strong over the luminosity range probed (-23.0<M_r < -18.0),but for red galaxies the dependence is weaker and becomes insignificant for luminosities below L^*. To examine whether a significant stochastic/nonlinear component exists in the bias relation, we study the ratio R_ij= W_{ii}W_{jj}/W_{ij}^2, where W_{ij} is the projected correlation between subsample i and j. We find that the values of R_ij are all consistent with 1 for all-all, red-red and blue-blue samples, however significantly larger than 1 for red-blue samples. For faint red - faint blue samples the values of R_{ij} are as high as ~ 2 on small scales r_p < 1 \mpch and decrease with increasing r_p.Comment: 25 pages, 18 figures, Accepted for publication in Ap

Assessing rotation-invariant feature classification for automated wildebeest population counts

Accurate and on-demand animal population counts are the holy grail for wildlife conservation organizations throughout the world because they enable fast and responsive adaptive management policies. While the collection of image data from camera traps, satellites, and manned or unmanned aircraft has advanced significantly, the detection and identification of animals within images remains a major bottleneck since counting is primarily conducted by dedicated enumerators or citizen scientists. Recent developments in the field of computer vision suggest a potential resolution to this issue through the use of rotation-invariant object descriptors combined with machine learning algorithms. Here we implement an algorithm to detect and count wildebeest from aerial images collected in the Serengeti National Park in 2009 as part of the biennial wildebeest count. We find that the per image error rates are greater than, but comparable to, two separate human counts. For the total count, the algorithm is more accurate than both manual counts, suggesting that human counters have a tendency to systematically over or under count images. While the accuracy of the algorithm is not yet at an acceptable level for fully automatic counts, our results show this method is a promising avenue for further research and we highlight specific areas where future research should focus in order to develop fast and accurate enumeration of aerial count data. If combined with a bespoke image collection protocol, this approach may yield a fully automated wildebeest count in the near future.CJT is supported by a Complex Systems Scholar Award from the James S. McDonnell Foundation. JGCH is supported by a Lord Kelvin Adam Smith Fellowship, funding from the British Ecological Society and the European Union’s Horizon 2020 research and innovation programme under grant agreement No 641918 AfricanBioServices. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Activity of raltitrexed and gemcitabine in advanced pancreatic cancer

Background: Gemcitabine has evolved as standard therapy in advanced pancreatic cancer since the demonstration of a significant clinical benefit. Phase II trials have shown that gemcitabine can be successfully combined with thymidylate synthase (TS) inhibitors such as continuous-infusion 5-fluorouracil (5-FU). However, continuous-infusion 5-FU is inconvenient because of the need for a central venous access. The aim of this study was to assess the efficacy and safety of gemcitabine in combination with raltitrexed (Tomudex), a novel and selective TS inhibitor that has the advantage of a 3-weekly treatment interval and manageable toxicity. Patients and methods: Chemotherapy-naïve patients with measurable advanced pancreatic cancer were treated with raltitrexed 3 mg/m2 as a 15-min infusion on day 1 and gemcitabine 1000 mg/m2 on days 1 and 8, every 21 days. Results: Twenty-five eligible patients (17 male, eight female) with metastatic (21 patients) or locally advanced (four patients) disease entered the study. The median number of courses per patient was four (range 1-14). One patient was not evaluable for response. There were three partial remissions [12%; 95% confidence interval (CI) 2.6% to 31.2%] and nine stable disease situations (36%; 95% CI 18.0% to 57.5%), while the tumours of 12 patients (48%; 95% CI 27.8% to 68.7%) showed progressive disease after three treatment cycles. WHO grade 3/4 toxicity was rare and symptomatic in only one patient, who experienced grade 4 diarrhoea and grade 3 nausea and vomiting. Symptomatic benefit was seen in 12 patients. Median survival was 185 days (95% CI 129-241) with six patients still alive. Conclusions: The efficacy of raltitrexed plus gemcitabine is limited, but compares well with other chemotherapy treatment options in advanced pancreatic cancer. However, this combination is convenient and symptomatic toxicity is rare. Thus, raltitrexed and gemcitabine should be investigated further in combination with drugs interfering with specific molecular target

CVAK104 is a Novel Regulator of Clathrin-mediated SNARE Sorting

Clathrin-coated vesicles (CCVs) mediate transport between the plasma membrane, endosomes and the trans Golgi network. Using comparative proteomics, we have identified coated-vesicle-associated kinase of 104 kDa (CVAK104) as a candidate accessory protein for CCV-mediated trafficking. Here, we demonstrate that the protein colocalizes with clathrin and adaptor protein-1 (AP-1), and that it is associated with a transferrin-positive endosomal compartment. Consistent with these observations, clathrin as well as the cargo adaptors AP-1 and epsinR can be coimmunoprecipitated with CVAK104. Small interfering RNA (siRNA) knockdown of CVAK104 in HeLa cells results in selective loss of the SNARE proteins syntaxin 8 and vti1b from CCVs. Morpholino-mediated knockdown of CVAK104 in Xenopus tropicalis causes severe developmental defects, including a bent body axis and ventral oedema. Thus, CVAK104 is an evolutionarily conserved protein involved in SNARE sorting that is essential for normal embryonic development

Untersuchungen über die Kallusbildung von di- und tetraploiden Reben in vitro

An Sproßexplantaten von di- und tetraploidem Vitis vinifera „Riesling" und „Portugieser" wurde das Kalluswachstum in vitro untersucht. Nach 42 Tagen hatte sich an den tetraploiden Explantaten der Sorte Riesling 28%, an den tetraploiden Explantaten der Sorte Portugieser 65% mehr Kallus gebildet als an den betreffenden diploiden Explantaten.Das höhere Frischgewicht der Kalli von tetraploiden Explantaten kann auf den höheren Wassergehalt der Zellen zurückgeführt werden. Es konnte kein Unterschied im Trockengewicht der Kalli von di- und tetraploiden Explantaten der Sorte Portugieser festgestellt werden. Die durchschnittliche Länge der Kalluszellen von tetraploiden Explantaten war gegenüber denen von diploiden Explantaten nahezu verdoppelt, während die Breite der Zellen gleich war. Dadurch war der Längen/Breitenindex bei den Kalluszellen der tetraploiden Explantate von 1,9 auf 3,3 erhöht. Das durchschnittliche Volumen der Kalluszellen der tetraploiden Explantate war verdoppelt.Die erhöhte Kallusbildung an den tetraploiden Explantaten muß zu einem gewissen Teil auf Polyploidisierungen zurückgeführt werden, die in beiden Kallustypen festgestellt werden konnte. Obwohl diese Polyploidisierungen in den Kalli der di- und tetraploiden Explantate gleich häufig auftraten, wirkte sich die dadurch bedingte sekundäre Vergrößerung der Zellen in den Kalli der tetraploiden Explantate stärker aus als in den Kalli der diploiden Explantate.Studies on callus growth of di- and tetraploid grapes in vitroCallus growth on shoot explants of di- and tetraploid Vitis vinifera "Riesling" and "Portugieser" was investigated in vitro. After 42 days the 4x explants showed on "Riesling" 28°/o more callus, and on "Portugieser" the increase was 65%. There was no difference in the dry weight of the calli of diploid and tetraploid "Portugieser" explants. Therefore, the increased fresh weight of the calli of the 4x explants can be traced back to a higher water content of the cells. The average length of the calli cells of the tetraploid explants was almost double that of the 2x, while the width was equal. Accordingly, the length/width index was increased from 1.9 to 3.3. The average volume of the callus cells of the 4x explants was doubled. The increased production of callus on the tetraploid explants must, to a certain extent, be traced back to polyploidisation, which is found equally in both types of callus. Although there is an equal occurence of polyploidisation in the calli of 2x and 4x explants, the secondary increase of cell size, which is due to this polyploidisation, is more prominent in the calli of the 4x explants

Towards cost-effective watershed management in the Brazilian Atlantic Forest: valuing forest ecosystem services related to water quality in the Guapi-Macacu watershed.

The Brazilian Atlantic forest is considered a global biodiversity hotspot, harbouring a large number of endemic species, and providing essential watershed services to millions of urban water users. However, agricultural expansion and urban development have reduced the forest area to less than 8% of its original size. Multiple pressures on the Atlantic forest ecosystem continue. Payments for ecosystem services (PES) schemes are increasingly proposed by local initiatives and supported by international cooperation to manage the trade‐offs among agricultural uses and watershed services in a cost‐effective manner. Studying the provision costs of as well as the demand for watershed services can help decision makers to evaluate the scope and economic feasibility of PES and alternative management options. The Guapi‐Macacu watershed in the state of Rio de Janeiro supplies water to 2.5 million inhabitants within five municipalities. Water resources are of utmost importance for agriculture and industries, such water bottle companies, breweries and the biggest Brazilian petrochemical complex COMPERJ. Our study concentrates on valuing watershed services in terms of controlling nutrient and sediment loads under different land use systems. Nutrient loads include Nitrogen and Phosphorus found in surface water, whereas sediment loads are measured in terms of turbidity and total solids. Replacement and avoided cost methods are applied focusing on the local water treatment facility to estimate demand for service maintenance and improvement. To provide empirical evidence of the link between land use/land cover and water quality indicators, we rely on water quality modelling and monitoring specifically adapted to this area. This takes place within the framework of the multi‐disciplinary German‐Brazilian cooperation project DINARIO/MP2. Spatial conservation opportunity cost analysis is used to study the costs of watershed services maintenance and improvement and derive implication for cost‐effective management on the basis of scenario analyses

Scaling properties of the redshift power spectrum: theoretical models

We report the results of an analysis of the redshift power spectrum $P^S(k,\mu)$ in three typical Cold Dark Matter (CDM) cosmological models, where $\mu$ is the cosine of the angle between the wave vector and the line-of-sight. Two distinct biased tracers derived from the primordial density peaks of Bardeen et al. and the cluster-underweight model of Jing, Mo, & B\"orner are considered in addition to the pure dark matter models. Based on a large set of high resolution simulations, we have measured the redshift power spectrum for the three tracers from the linear to the nonlinear regime. We investigate the validity of the relation - guessed from linear theory - in the nonlinear regime $$P^S(k,\mu)=P^R(k)[1+\beta\mu^2]^2D(k,\mu,\sigma_{12}(k)),$$ where $P^R(k)$ is the real space power spectrum, and $\beta$ equals $\Omega_0^{0.6}/b_l$. The damping function $D$ which should generally depend on $k$, $\mu$, and $\sigma_{12}(k)$, is found to be a function of only one variable $k\mu\sigma_{12}(k)$. This scaling behavior extends into the nonlinear regime, while $D$ can be accurately expressed as a Lorentz function - well known from linear theory - for values $D > 0.1$. The difference between $\sigma_{12}(k)$ and the pairwise velocity dispersion defined by the 3-D peculiar velocity of the simulations (taking $r=1/k$) is about 15%. Therefore $\sigma_{12}(k)$ is a good indicator of the pairwise velocity dispersion. The exact functional form of $D$ depends on the cosmological model and on the bias scheme. We have given an accurate fitting formula for the functional form of $D$ for the models studied.Comment: accepted for publication in ApJ;24 pages with 7 figures include