Large-Area Two-Dimensional Plasmonic Meta-Glasses and Meta-Crystals: a Comparative Study

The geometrical arrangement of metallic nanoparticles plays a crucial role on the optical response of nanoplasmonic samples due to particle-particle interactions. In this work, large-area, two-dimensional meta-glasses (random arrangements) and meta-crystals (periodic arrangements) made of identical metallic nanoparticles are investigated for three different particle densities of 5, 10, and 15 discs/μm². A direct comparison between random and periodically ordered arrays is presented. The comparison clearly shows that the particle density has the largest influence on the extinction spectra for both periodic and random samples, and that for equal densities, the optical response away from diffraction effects is strikingly similar in both cases. The role of the radial density function and minimum particle distance is also determined. This study elucidates the role of the particle-particle interactions on the response of plasmonic nanoparticles and indicates how to control position and shape of the plasmonic resonance.     

A generic high-throughput screening assay for kinases: protein kinase a as an example

A generic high-throughput screening assay based on the scintillation proximity assay technology has been developed for protein kinases. In this assay, the biotinylated (33)P-peptide product is captured onto polylysine Ysi bead via avidin. The scintillation signal measuring the product formation increases linearly with avidin concentration due to effective capture of the product on the bead surface via strong coulombic interactions. This novel assay has been optimized and validated in 384-well microplates. In a pilot screen, a signal-to-noise ratio of 5- to 9-fold and a Z' factor ranging from 0.6 to 0.8 were observed, demonstrating the suitability of this assay for high-throughput screening of random chemical libraries for kinase inhibitors.     

Suitability of Nicotinic Acetylcholine Receptor α7 and Muscarinic Acetylcholine Receptor 3 Antibodies for Immune Detection: Evaluation in Murine Skin

Recent evidence reveals a crucial role for acetylcholine and its receptors in the regulation of inflammation, particularly of nicotinic acetylcholine receptor α7 (Chrna7) and muscarinic acetylcholine receptor 3 (Chrm3). Immunohistochemistry is a key tool for their cellular localization in functional tissues. We evaluated nine different commercially available antibodies on back skin tissue from wild-type (Wt) and gene-deficient (KO) mice. In the immunohistochemical analysis, we focused on key AChR-ligand sensitive skin cells (mast cells, nerve fibers and keratinocytes). All five antibodies tested for Chrm3 and the first three Chrna7 antibodies stained positive in both Wt and respective KO skin. With the 4th antibody (ab23832) nerve fibers were unlabeled in the KO mice. By western blot analysis, this antibody detected bands in both Wt and Chrna7 KO skin and brain. qRT-PCR revealed mRNA amplification with a primer set for the undeleted region in both Wt and KO mice, but none with a primer set for the deleted region in KO mice. By 2D electrophoresis, we found β-actin and β-enolase cross reactivity, which was confirmed by double immunolabeling. In view of the present results, the tested antibodies are not suitable for immunolocalization in skin and suggest thorough control of antibody specificity is required if histomorphometry is intended.     

Domestication Shapes Recombination Patterns in Tomato

Meiotic recombination is a biological process of key importance in breeding, to generate genetic diversity and develop novel or agronomically relevant haplotypes. In crop tomato, recombination is curtailed as manifested by linkage disequilibrium decay over a longer distance and reduced diversity compared with wild relatives. Here, we compared domesticated and wild populations of tomato and found an overall conserved recombination landscape, with local changes in effective recombination rate in specific genomic regions. We also studied the dynamics of recombination hotspots resulting from domestication and found that loss of such hotspots is associated with selective sweeps, most notably in the pericentromeric heterochromatin. We detected footprints of genetic changes and structural variants, among them associated with transposable elements, linked with hotspot divergence during domestication, likely causing fine-scale alterations to recombination patterns and resulting in linkage drag.     

Reaction Mechanism of the Bicopper Enzyme Peptidylglycine α-Hydroxylating Monooxygenase

Peptidylglycine α-hydroxylating monooxygenase is a noninteracting bicopper enzyme that stereospecifically hydroxylates the terminal glycine of small peptides for its later amidation. Neuroendocrine messengers, such as oxytocin, rely on the biological activity of this enzyme. Each catalytic turnover requires one oxygen molecule, two protons from the solvent, and two electrons. Despite this enzyme having been widely studied, a consensus on the reaction mechanism has not yet been found. Experiments and theoretical studies favor a pro-S abstraction of a hydrogen atom followed by the rebinding of an OH group. However, several hydrogen-abstracting species have been postulated; because two protons are consumed during the reaction, several protonation states are available. An electron transfer between the copper atoms could play a crucial role for the catalysis as well. This leads to six possible abstracting species. In this study, we compare them on equal footing. We perform quantum mechanics/molecular mechanics calculations, considering the glycine hydrogen abstraction. Our results suggest that the most likely mechanism is a protonation of the abstracting species before the hydrogen abstraction and another protonation as well as a reduction before OH rebinding.     

Round versus flat: bone cell morphology, elasticity, and mechanosensing

There is increasing evidence that cell function and mechanical properties are closely related to morphology. However, most in vitro studies investigate flat adherent cells, which might not reflect physiological geometries in vivo. Osteocytes, the mechanosensors in bone, reside within ellipsoid containment, while osteoblasts adhere to flatter bone surfaces. It is unknown whether morphology difference, dictated by the geometry of attachment is important for cell rheology and mechanosensing. We developed a novel methodology for investigating the rheology and mechanosensitivity of bone cells under different morphologies using atomic force microscopy and our two-particle assay for optical tweezers. We found that the elastic constant of MLO-Y4 osteocytes when flat and adherent (>1 kPa) largely differed when round but partially adherent (<1 kPa). The elastic constant of round suspended MLO-Y4 osteocytes, MC3T3-E1 osteoblasts, and primary osteoblasts were similarly <1 kPa. The mechanosensitivity of round suspended MLO-Y4 osteocytes was investigated by monitoring nitric oxide (NO) release, an essential signaling molecule in bone. A preliminary observation of high NO release from round suspended MLO-Y4 osteocytes in response to 5 pN force is reported here, in contrast with previous studies where flat cells routinely release lesser NO while being stimulated with higher force. Our results suggest that a round cellular morphology supports a less stiff cytoskeleton configuration compared with flat cellular morphology. This implies that osteocytes take advantage of their ellipsoid morphology in vivo to sense small strains benefiting bone health. Our assay provides novel opportunities for in vitro studies under a controlled suspended morphology versus commonly studied adherent morphologies.     

Microbial metabolism of quinoline and related compounds. V. Degradation of 1H-4-oxoquinoline by Pseudomonas putida 33/1

A bacterial strain was isolated with the ability to use 1H-4-oxoquinoline as the sole source of carbon, nitrogen and energy. On the basis of its physiological properties, this isolate was classified as Pseudomonas putida. 1H-3-Hydroxy-4-oxoquinoline, N-formylanthranilic acid, anthranilic acid and catechol were identified as intermediates in the degradation pathway. The latter was further degraded by ortho-cleavage. The enzymatic conversion of 1H-4-oxoquinoline into 1H-3-hydroxy-4-oxoquinoline requires oxygen and NADH. Experiments with 18O2 showed that the oxygen consumed in this enzymatic reaction is derived from the atmosphere.