A native chemical ligation approach for combinatorial assembly of affibody molecules

Affinity molecules labeled with different reporter groups, such as fluorophores or radionuclides, are valuable research tools used in a variety of applications. One class of engineered affinity proteins is Affibody molecules, which are small (6.5 kDa) proteins that can be produced by solid phase peptide synthesis (SPPS), thereby allowing site-specific incorporation of reporter groups during synthesis. The Affibody molecules are triple-helix proteins composed of a variable part, which gives the protein its binding specificity, and a constant part, which is identical for all Affibody molecules. In the present study, native chemical ligation (NCL) has been applied for combinatorial assembly of Affibody molecules from peptide fragments produced by Fmoc SPPS. The concept is demonstrated for the synthesis of three different Affibody molecules. The cysteine residue introduced at the site of ligation can be used for directed immobilization and does not interfere with the function of the investigated proteins. This strategy combines a high-yield production method with facilitated preparation of proteins with different C-terminal modifications.     

Loss aversion and price volatility as determinants of attitude towards and preference for variable price in the Swedish electricity market

The results of a survey of a random sample of 488 Swedish residents showed that a positive attitude towards and preference for a variable price agreement with the incumbent electricity supplier was negatively affected by loss aversion, and a positive attitude also negatively affected by beliefs about price volatility. Although correlated with attitude and preference, age, education, and current choice of a variable price agreement had no independent effects. Income and current electricity costs had no effects.     

Interactions of Skin with Gold Nanoparticles of Different Surface Charge,Shape, and Functionality

The interactions between skin and colloidal gold nanoparticles of different physicochemical characteristics are investigated. By systematically varying the charge, shape, and functionality of gold nanoparticles, the nanoparticle penetration through the different skin layers is assessed. The penetration is evaluated both qualitatively and quantitatively using a variety of complementary techniques. Inductively coupled plasma optical emission spectrometry (ICP‐OES) is used to quantify the total number of particles which penetrate the skin structure. Transmission electron microscopy (TEM) and two photon photoluminescence microscopy (TPPL) on skin cross sections provide a direct visualization of nanoparticle migration within the different skin substructures. These studies reveal that gold nanoparticles functionalized with cell penetrating peptides (CPPs) TAT and R₇ are found in the skin in larger quantities than polyethylene glycol‐functionalized nanoparticles, and are able to enter deep into the skin structure. The systematic studies presented in this work may be of strong interest for developments in transdermal administration of drugs and therapy.     

Photonic crystal patterning of luminescent sol-gel films for light extraction

Structured luminescent thin films are investigated in the context of improved light extraction of phosphors for solid-state-lighting applications. Thin films composed of a sol-gel titania matrix doped with europium chelates are studied as a model system. These films, patterned with a square photonic lattice by soft nanoimprint lithography, are characterized by angle-resolved fluorescence. Modeling of this simple technique is shown to fit well the experimental data, revealing in great detail the guided modes of the film and their extraction parameters. An eightfold extraction enhancement factor of the film emission is measured. To further improve the extraction efficiency, we investigate the role of an additional low-index mesoporous silica underlayer through its influence on the guided modes of different polarizations and their interactions with the photonic crystal. Results obtained on model systems open the way towards the optimization of light-emitting devices, using a strategy of dielectric microstructure engineering using the sol-gel process.     

Cyclic volatile methylsiloxane bioaccumulation in flounder and ragworm in the Humber Estuary

Cyclic volatile methylsiloxanes are being subjected to regulatory scrutiny as possible PBT chemicals. The investigation of bioaccumulation has yielded apparently contradictory results, with high laboratory fish bioconcentration factors on the one hand and low field trophic magnification factors on the other. In this study, octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), and dodecamethylcyclohexasiloxane (D6) were studied along with polychlorinated biphenyls (PCBs) in sediments, ragworm, and flounder from six sites in the Humber Estuary. Bioaccumulation was evaluated using multimedia bioaccumulation factors (mmBAFs) which quantified the fraction of the contaminant present in the aquatic environment that is transferred to the biota. PCB 180, a known strongly bioaccumulative chemical, was used as a benchmark. The mean mmBAF of D5 was about twice that of PCB 180 in both polycheates and flounder, while for D4 it was 6 and 14 times higher, respectively. The mmBAF of D6 was a factor 5-10 lower than that of PCB180. The comparatively strong multimedia bioaccumulation of D4 and D5, even in the absence of biomagnification, was explained by both compounds having a >100 times stronger tendency to partition into lipid rather than into organic carbon, while PCB 180 partitions to a similar extent into both matrices.     

Robustness and sensitivity of a blueprint for on-farm estimation of dairy cow energy balance

Excessive negative energy balance (EB) has been associated with decreased reproductive performance and increased risk of lameness and metabolic diseases. On-farm, automated EB estimates for individual cows would enable dairy farmers to detect excessive negative EB early and act to minimize its extent and duration by altering feeding. Previously, we have shown that EB can be estimated from frequent measurements of body weight (BW) and body condition score (BCS) changes, referred to as EB_body. In this study, we investigated the robustness and sensitivity of the EB_body method to assess its genericity and on-farm applicability. We used 5 data sets with BW of lactating cows (name of data set in parenthesis): 65 Holstein cows in a French feeding trial (INRA); 6 Holstein cows in a British feeding trial (Friggens); 31 Holstein cows and 17 Jersey cows in a Danish feeding trial (DCRC); 140 Holstein cows in a British feeding trial (Scotland's Rural College, SRUC); and 1,592 Holstein cows on 9 Danish farms with milking robots (automatic milking system). We used the INRA and Friggens data sets to develop a dynamic formula to correct BW for increasing residual gut-fill (RGF) during early lactation. With the DCRC data, we tested the effect of smoothing parameters and weighing frequency on EB_body. Also, 2 robustness tests were performed using the SRUC data to test the effect of diet change on BW and the automatic milking system data to test the effect of farm on BW variation. Finally, we combined the results into a blueprint describing different ways to calculate EB_body depending on the purpose and on the availability of BCS. The dynamic RGF adjustment resulted in a lower empty BW during early lactation than that obtained with the previously used constant RGF. The double-exponential smoothing method used to correct for meal-related gut-fill was robust to choice of smoothing parameters. Cows should be weighed at least once every 4 d during early lactation to capture the duration of negative EB_body. Our EB_body method proved robust to diet changes. Finally, although cow BW varied significantly between farms, the quantile regression smoothing of BW did not bias the estimation of weight differences between herds. In conclusion, these results validate the applicability of the EB_body method to estimate EB across a range of farm conditions, and we provided a blueprint that enables the estimation of EB_body for individual cows on-farm using only frequent BW, in combination with BCS when available.     

Site‐Specific Antibody Labeling by Covalent Photoconjugation of Z Domains Functionalized for Alkyne–Azide Cycloaddition Reactions

Antibodies are extensively used in research, diagnostics, and therapy, and for many applications the antibodies need to be labeled. Labeling is typically performed by using amine‐reactive probes that target surface‐exposed lysine residues, resulting in heterogeneously labeled antibodies. An alternative labeling strategy is based on the immunoglobulin G (IgG)‐binding protein domain Z, which binds to the Fc region of IgG. Introducing the photoactivable amino acid benzoylphenylalanine (BPA) into the Z domain makes it possible for a covalent bond to be be formed between the Z domain and the antibody on UV irradiation, to produce a site‐specifically labeled product. Z₃₂BPA was synthesized by solid‐phase peptide synthesis and further functionalized to give alkyne‐Z₃₂BPA and azide‐Z₃₂BPA for Cu^I‐catalyzed cycloaddition, as well as DBCO‐Z₃₂BPA for Cu‐free strain‐promoted cycloaddition. The Z₃₂BPA variants were conjugated to the human IgG1 antibody trastuzumab and site‐specifically labeled with biotin or fluorescein. The fluorescently labeled trastuzumab showed specific staining of the membranes of HER2‐expressing cells in immunofluorescence microscopy.     

Does contact reduce prejudice or does prejudice reduce contact? A longitudinal test of the contact hypothesis among majority and minority groups in three european countries.

A widely researched panacea for reducing intergroup prejudice is the contact hypothesis. However, few longitudinal studies can shed light on the direction of causal processes: from contact to prejudice reduction (contact effects) or from prejudice to contact reduction (prejudice effects). The authors conducted a longitudinal field survey in Germany, Belgium, and England with school students. The sample comprised members of both ethnic minorities (n = 512) and ethnic majorities (n = 1,143). Path analyses yielded both lagged contact effects and prejudice effects: Contact reduced prejudice, but prejudice also reduced contact. Furthermore, contact effects were negligible for minority members. These effects were obtained for 2 indicators of prejudice: negative intergroup emotions and desire for social distance. For both majority and minority members, contact effects on negative emotions were stronger when outgroup contacts were perceived as being typical of their group. Contact effects were also mediated by intergroup anxiety. This mediating mechanism was impaired for minority members because of a weakened effect of anxiety on desire for social distance. Theoretical and practical implications of these findings are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Phosphorylation but Not Oligomerization Drives the Accumulation of Tau with Nucleoporin Nup98

Tau is a neuronal protein that stabilizes axonal microtubules (MTs) in the central nervous system. In Alzheimer’s disease (AD) and other tauopathies, phosphorylated Tau accumulates in intracellular aggregates, a pathological hallmark of these diseases. However, the chronological order of pathological changes in Tau prior to its cytosolic aggregation remains unresolved. These include its phosphorylation and detachment from MTs, mislocalization into the somatodendritic compartment, and oligomerization in the cytosol. Recently, we showed that Tau can interact with phenylalanine-glycine (FG)-rich nucleoporins (Nups), including Nup98, that form a diffusion barrier inside nuclear pore complexes (NPCs), leading to defects in nucleocytoplasmic transport. Here, we used surface plasmon resonance (SPR) and bio-layer interferometry (BLI) to investigate the molecular details of Tau:Nup98 interactions and determined how Tau phosphorylation and oligomerization impact the interactions. Importantly, phosphorylation, but not acetylation, strongly facilitates the accumulation of Tau with Nup98. Oligomerization, however, seems to inhibit Tau:Nup98 interactions, suggesting that Tau-FG Nup interactions occur prior to oligomerization. Overall, these results provide fundamental insights into the molecular mechanisms of Tau-FG Nup interactions within NPCs, which might explain how stress-and disease-associated posttranslational modifications (PTMs) may lead to Tau-induced nucleocytoplasmic transport (NCT) failure. Intervention strategies that could rescue Tau-induced NCT failure in AD and tauopathies will be further discussed.