TY - JOUR AB - From a simple thought to a multicellular movement AU - Amberg, Nicole AU - Stouffer, Melissa A AU - Vercellino, Irene ID - 12282 IS - 8 JF - Journal of Cell Science SN - 0021-9533 TI - Operation STEM fatale – how an equity, diversity and inclusion initiative has brought us to reflect on the current challenges in cell biology and science as a whole VL - 135 ER - TY - JOUR AB - Mica-titania pearlescent pigments (MTs) were previously coated with organic molecules to obtain combination pigments (CPs) for achieving certain improvements or functionalities. Anthocyanins (ACNs) are molecules that can be extracted from natural resources and exhibit color changes via pH modifications of the enclosing medium. The purpose of the study was to produce a new series of CPs by depositing ACNs on MTs at different pH values, to observe the changes in color, and to associate these changes to thermogravimetrically determined deposition efficiencies in light of spectral differences. The extraction and deposition methods were based on aqueous chemistry and were straightforward. The ACN deposition generally increased with increasing pH and correlated with the consistency between the charges of the MT surfaces and the dominant ACN species at a specific pH value. The fluorescence of the CPs was inversely correlated with the deposition quantities invoking the possibility of a quenching effect. AU - Çoruh, Mehmet Orkun AU - Gündüz, Güngör AU - Çolak, Üner AU - Maviş, Bora ID - 10945 IS - 2 JF - Colorants SN - 2079-6447 TI - pH-dependent coloring of combination effect pigments with anthocyanins from Brassica oleracea var. capitata F. rubra VL - 1 ER - TY - JOUR AB - Nanobodies (VHH) from camelid antibody libraries hold great promise as therapeutic agents and components of immunoassay systems. Synthetic antibody libraries that could be designed and generated once and for various applications could yield binders to virtually any targets, even for non-immunogenic or toxic ones, in a short term. One of the most difficult tasks is to obtain antibodies with a high affinity and specificity to polyglycosylated proteins. It requires antibody libraries with extremely high functional diversity and the use of sophisticated selection techniques. Here we report a development of a novel sandwich immunoassay involving a combination of the synthetic library-derived VHH-Fc fusion protein as a capture antibody and the immune single-chain fragment variable (scFv) as a tracer for the detection of pregnancy-associated glycoprotein (PAG) of cattle (Bos taurus). We succeeded in the generation of a number of specific scFv antibodies against PAG from the mouse immune library. Subsequent selection using the immobilized scFv-Fc capture antibody allowed to isolate 1.9 nM VHH binder from the diverse synthetic library without any overlapping with the capture antibody binding site. The prototype sandwich ELISA based on the synthetic VHH and the immune scFv was established. This is the first successful example of the combination of synthetic and immune antibody libraries in a single sandwich immunoassay. Thus, our approach could be used for the express isolation of antibody pairs and the development of sandwich immunoassays for challenging antigens. AU - Dormeshkin, Dmitri AU - Shapira, Michail AU - Karputs, Alena AU - Kavaleuski, Anton AU - Kuzminski, Ivan AU - Stepanova, Elena AU - Gilep, Andrei ID - 11462 JF - Applied Microbiology and Biotechnology SN - 0175-7598 TI - Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development VL - 106 ER - TY - JOUR AB - N-1-naphthylphthalamic acid (NPA) is a key inhibitor of directional (polar) transport of the hormone auxin in plants. For decades, it has been a pivotal tool in elucidating the unique polar auxin transport-based processes underlying plant growth and development. Its exact mode of action has long been sought after and is still being debated, with prevailing mechanistic schemes describing only indirect connections between NPA and the main transporters responsible for directional transport, namely PIN auxin exporters. Here we present data supporting a model in which NPA associates with PINs in a more direct manner than hitherto postulated. We show that NPA inhibits PIN activity in a heterologous oocyte system and that expression of NPA-sensitive PINs in plant, yeast, and oocyte membranes leads to specific saturable NPA binding. We thus propose that PINs are a bona fide NPA target. This offers a straightforward molecular basis for NPA inhibition of PIN-dependent auxin transport and a logical parsimonious explanation for the known physiological effects of NPA on plant growth, as well as an alternative hypothesis to interpret past and future results. We also introduce PIN dimerization and describe an effect of NPA on this, suggesting that NPA binding could be exploited to gain insights into structural aspects of PINs related to their transport mechanism. AU - Abas, Lindy AU - Kolb, Martina AU - Stadlmann, Johannes AU - Janacek, Dorina P. AU - Lukic, Kristina AU - Schwechheimer, Claus AU - Sazanov, Leonid A AU - Mach, Lukas AU - Friml, Jiří AU - Hammes, Ulrich Z. ID - 8993 IS - 1 JF - PNAS SN - 00278424 TI - Naphthylphthalamic acid associates with and inhibits PIN auxin transporters VL - 118 ER - TY - JOUR AB - Cryo-EM grid preparation is an important bottleneck in protein structure determination, especially for membrane proteins, typically requiring screening of a large number of conditions. We systematically investigated the effects of buffer components, blotting conditions and grid types on the outcome of grid preparation of five different membrane protein samples. Aggregation was the most common type of problem which was addressed by changing detergents, salt concentration or reconstitution of proteins into nanodiscs or amphipols. We show that the optimal concentration of detergent is between 0.05 and 0.4% and that the presence of a low concentration of detergent with a high critical micellar concentration protects the proteins from denaturation at the air-water interface. Furthermore, we discuss the strategies for achieving an adequate ice thickness, particle coverage and orientation distribution on free ice and on support films. Our findings provide a clear roadmap for comprehensive screening of conditions for cryo-EM grid preparation of membrane proteins. AU - Kampjut, Domen AU - Steiner, Julia AU - Sazanov, Leonid A ID - 9205 IS - 3 JF - iScience TI - Cryo-EM grid optimization for membrane proteins VL - 24 ER -