Convenient Method of Micrometer-Scale Excitation of Propagating Surface Plasmons by a Focused Laser Beam

Plasmonics - Surface plasmons (SPs) excited on microscopic areas are desired in a variety of applications, including SP microscopy and (bio)sensing. We present an optical method of excitation of...     

Protein τ-Mediated Effects on Rat Hippocampal Choline Transporters CHT1 and τ-Amyloid β Interactions

It is suggested that intracellular tau protein (τ), when released extracellularly upon neuron degeneration, could evoke direct toxic effects on the cholinergic neurotransmitter system through muscarinic receptors and thus contribute to the pathogenesis of Alzheimer’s disease. In this study, we evaluated the in vitro effects of six naturally occurring monomeric τ isoforms on rat hippocampal synaptosomal choline transporters CHT1 (large transmembrane proteins associated with high-affinity choline transport and vulnerable to actions of amyloid β peptides (Aβ) applied in vitro or in vivo). Some τ isoforms at nM concentrations inhibited choline transport in a dose- and time-dependent saturable manner (352 = 441 > 410 = 383 > 381 = 412) and effects were associated with changes in the Michaelis constant rather than in maximal velocity. Moreover, the actions of τ 352/441 were not influenced by previous depolarisation of synaptosomes or by previous depletion of membrane cholesterol. Specific binding of [3H]hemicholinium-3 was not significantly altered by τ 352/441 at higher nM concentrations. Results of in vitro tests on CHT1 transporters from cholesterol-depleted synaptosomes supported interactions between Aβ 1-40 and τ 352. In addition, we developed surface plasmon resonance biosensors to monitor complexes of Aβ 1-42 and τ 352 using a sandwich detection format. It seems, therefore, that protein τ, similar to Aβ peptides, can contribute to the pathogenesis of Alzheimer’s disease through its actions on CHT1 transporters. However, the interaction mechanisms are quite different (τ probably exerts its effects through direct interactions of microtubule binding repeats with extracellular portions of the CHT1 protein without influencing the choline recognition site, Aβ rather through lipid rafts in the surrounding membranes). An N-terminal insert of τ is not necessary but the N-terminal projection domain plays a role. The developed biosensor will be used to detect Aβ–τ complexes in cerebrospinal fluid in order to evaluate them as prospective biomarkers of Alzheimer′s disease.     

Compact and low-cost biosensor based on novel approach to spectroscopy of surface plasmons

A high-performance surface plasmon resonance (SPR) sensor based on a novel approach to spectroscopy of surface plasmons is reported. This approach employs a special diffraction grating structure (referred to as surface plasmon resonance coupler and disperser, SPRCD) which simultaneously couples light into a surface plasmon and disperses the diffracted light for spectral readout of SPR signal. The developed SPRCD sensor consists of a miniature cartridge integrating the diffraction grating and microfluidics and a compact optical system which simultaneously acquires data from four independent sensing channels in the cartridge. It is demonstrated that the SPRCD sensor is able to measure bulk refractive index changes as small as 3 × 10⁻⁷ RIU (refractive index units) and to detect short oligonucleotides in concentrations down to 200 pM.     

Human interleukin‐23 receptor antagonists derived from an albumin‐binding domain scaffold inhibit IL‐23‐dependent ex vivo expansion of IL‐17‐producing T‐cells

Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin‐23 receptor (IL‐23R), which is a key element of proinflammatory IL‐23‐mediated signaling. A library of variants derived from the three‐helix bundle scaffold of the albumin‐binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high‐affinity binders of recombinant extracellular IL‐23R. A collection of 34 IL‐23R‐binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL‐23, or the biologically active human IL‐23 cytokine, to the recombinant IL‐23R or soluble IL‐23R‐IgG chimera. The strongest competitors for IL‐23R binding in ELISA were confirmed to recognize human IL‐23R‐IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub‐ to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K‐562, THP‐1 and Jurkat, and this binding correlated with IL‐23R cell‐surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP‐1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL‐23‐driven expansion of IL‐17‐producing primary human CD4⁺ T‐cells. Thus, we conclude that unique IL‐23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti‐inflammatory biologicals. Proteins 2014; 82:975–989. © 2013 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Ambiguous Refractive Index Sensitivity of Fano Resonance on an Array of Gold Nanoparticles

We investigate the optical response to refractive index changes of a Fano resonance occurring in a random array of gold nanoparticles supported on a glass substrate. The Fano resonance results from the interference between localized surface plasmon on a gold nanoparticle and the light reflected at the boundary of the glass substrate. We demonstrate that the sensitivity of the resonance to the refractive index of the surrounding medium is highly dependent on the excitation geometry and can assume either positive or negative values. We furthermore present a theoretical analysis explaining this behavior based on the rigorous coupled wave analysis (RCWA) as well as the island film theory.

     

Interactions of 17β-Hydroxysteroid Dehydrogenase Type 10 and Cyclophilin D in Alzheimer's Disease

The nucleus-encoded 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) regulates cyclophilin D (cypD) in the mitochondrial matrix. CypD regulates opening of mitochondrial permeability transition pores. Both mechanisms may be affected by amyloid β peptides accumulated in mitochondria in Alzheimer's disease (AD). In order to clarify changes occurring in brain mitochondria, we evaluated interactions of both mitochondrial proteins in vitro (by surface plasmon resonance biosensor) and detected levels of various complexes of 17β-HSD10 formed in vivo (by sandwich ELISA) in brain mitochondria isolated from the transgenic animal model of AD (homozygous McGill-R-Thy1-APP rats) and in cerebrospinal fluid samples of AD patients. By surface plasmon resonance biosensor, we observed the interaction of 17β-HSD10 and cypD in a direct real-time manner and determined, for the first time, the kinetic parameters of the interaction (k_a 2.0?×?10⁵ M¹s^?1, k_d 5.8?×?10⁴ s^?1, and K_D 3.5?×?10^–10 M). In McGill-R-Thy1-APP rats compared to controls, levels of 17β-HSD10–cypD complexes were decreased and those of total amyloid β increased. Moreover, the levels of 17β-HSD10–cypD complexes were decreased in cerebrospinal fluid of individuals with AD (in mild cognitive impairment as well as dementia stages) or with Frontotemporal lobar degeneration (FTLD) compared to cognitively normal controls (the sensitivity of the complexes to AD dementia was 92.9%, that to FTLD 73.8%, the specificity to AD dementia equaled 91.7% in a comparison with the controls but only 26.2% with FTLD). Our results demonstrate the weakened ability of 17β-HSD10 to regulate cypD in the mitochondrial matrix probably via direct effects of amyloid β. Levels of 17β-HSD10–cypD complexes in cerebrospinal fluid seem to be the very sensitive indicator of mitochondrial dysfunction observed in neurodegeneration but unfortunately not specific to AD pathology. We do not recommend it as the new biomarker of AD.

     

A new surface plasmon resonance sensor for high-throughput screening applications

We report a new high-throughput surface plasmon resonance (SPR) sensor based on combination of SPR imaging with polarization contrast and a spatially patterned multilayer SPR structure. We demonstrate that this approach offers numerous advantageous features including high-contrast SPR images suitable for automated computer analysis, minimum crosstalk between neighboring sensing channels and inherent compensation for light level fluctuations. Applications of a laboratory prototype of the high-throughput SPR sensor with 108 sensing channels for refractometry and biosensing are described. In refractometric experiments, the noise-limited refractive index resolution of the system has been established to be 3 x 10(-6) refractive index unit (RIU). Experimental data on detection of human choriogonadotropin (hCG) suggest that in conjunction with monoclonal antibodies against hCG, the reported SPR imaging sensor is capable of detecting hCG at concentrations lower than 500 ng/ml.     

Multi-analyte surface plasmon resonance biosensing

Surface plasmon resonance (SPR) biosensors are affinity sensing devices exploiting a special mode of electromagnetic field-surface plasmon-polariton-to detect the binding of analyte molecules from a liquid sample to biomolecular recognition elements immobilized on the surface of the sensor. In this paper, we review advances of SPR biosensor technology towards detection systems for the simultaneous detection of multiple analytes (multi-analyte detection). In addition, we report application of a recently developed multichannel SPR sensor based on spectroscopy of surface plasmons and wavelength division multiplexing of sensing channels to multi-analyte detection.     

Drosophila Myt1 is a Cdk1 inhibitory kinase that regulates multiple aspects of cell cycle behavior during gametogenesis

The metazoan Wee1-like kinases Wee1 and Myt1 regulate the essential mitotic regulator Cdk1 by inhibitory phosphorylation. This regulatory mechanism, which prevents Cdk1 from triggering premature mitotic events, is also induced during the DNA damage response and used to coordinate cell proliferation with crucial developmental events. Despite the previously demonstrated role for Myt1 regulation of Cdk1 during meiosis, relatively little is known of how Myt1 functions at other developmental stages. To address this issue, we have undertaken a functional analysis of Drosophila Myt1 that has revealed novel developmental roles for this conserved cell cycle regulator during gametogenesis. Notably, more proliferating cells were observed in myt1 mutant testes and ovaries than controls. This can partly be attributed to ectopic division of germline-associated somatic cells in myt1 mutants, suggesting that Myt1 serves a role in regulating exit from the cell cycle. Moreover, mitotic index measurements suggested that germline stem cells proliferate more rapidly, in myt1 mutant females. In addition, male myt1 germline cells occasionally undergo an extra mitotic division, resulting in meiotic cysts with twice the normal numbers of cells. Based on these observations, we propose that Myt1 serves unique Cdk1 regulatory functions required for efficient coupling of cell differentiation with cell cycle progression.     

RAPTURE (RAD capture) panel facilitates analyses characterizing sea lamprey reproductive ecology and movement dynamics

Genomic tools are lacking for invasive and native populations of sea lamprey (Petromyzon marinus). Our objective was to discover single nucleotide polymorphism (SNP) loci to conduct pedigree analyses to quantify reproductive contributions of adult sea lampreys and dispersion of sibling larval sea lampreys of different ages in Great Lakes tributaries. Additional applications of data were explored using additional geographically expansive samples. We used restriction site‐associated DNA sequencing (RAD‐Seq) to discover genetic variation in Duffins Creek (DC), Ontario, Canada, and the St. Clair River (SCR), Michigan, USA. We subsequently developed RAD capture baits to genotype 3,446 RAD loci that contained 11,970 SNPs. Based on RAD capture assays, estimates of variance in SNP allele frequency among five Great Lakes tributary populations (mean F_ST 0.008; range 0.00–0.018) were concordant with previous microsatellite‐based studies; however, outlier loci were identified that contributed substantially to spatial population genetic structure. At finer scales within streams, simulations indicated that accuracy in genetic pedigree reconstruction was high when 200 or 500 independent loci were used, even in situations of high spawner abundance (e.g., 1,000 adults). Based on empirical collections of larval sea lamprey genotypes, we found that age‐1 and age‐2 families of full and half‐siblings were widely but nonrandomly distributed within stream reaches sampled. Using the genomic scale set of SNP loci developed in this study, biologists can rapidly genotype sea lamprey in non‐native and native ranges to investigate questions pertaining to population structuring and reproductive ecology at previously unattainable scales.