Selective extracellular vesicle-mediated export of an overlapping set of microRNAs from multiple cell types

Background: MicroRNAs (miRNAs) are a class of small RNA molecules that regulate expression of specific mRNA targets. They can be released from cells, often encapsulated within extracellular vesicles (EVs), and therefore have the potential to mediate intercellular communication. It has been suggested that certain miRNAs may be selectively exported, although the mechanism has yet to be identified. Manipulation of the miRNA content of EVs will be important for future therapeutic applications. We therefore wished to assess which endogenous miRNAs are enriched in EVs and how effectively an overexpressed miRNA would be exported.

Results: Small RNA libraries from HEK293T cells and vesicles before or after transfection with a vector for miR-146a overexpression were analysed by deep sequencing. A subset of miRNAs was found to be enriched in EVs; pathway analysis of their predicted target genes suggests a potential role in regulation of endocytosis. RT-qPCR in additional cell types and analysis of publicly available data revealed that many of these miRNAs tend to be widely preferentially exported. Whilst overexpressed miR-146a was highly enriched both in transfected cells and their EVs, the cellular:EV ratios of endogenous miRNAs were not grossly altered. MiR-451 was consistently the most highly exported miRNA in many different cell types. Intriguingly, Argonaute2 (Ago2) is required for miR-451 maturation and knock out of Ago2 has been shown to decrease expression of other preferentially exported miRNAs (eg miR-150 and miR-142-3p).

Conclusion: The global expression data provided by deep sequencing confirms that specific miRNAs are enriched in EVs released by HEK293T cells. Observation of similar patterns in a range of cell types suggests that a common mechanism for selective miRNA export may exist.

Multiple self-aligned iron nanowires by a dual selective chemical vapor deposition process

We have demonstrated a self-aligned process to fabricate organized iron nanowires on a planarized surface with wire dimensions down to 50 nm. Polishing was used to expose an alternating silicon silicon dioxide edge and a dual selective metal deposition process produced the nanowires. The initial selective deposition produced a tungsten layer on the exposed polysilicon regions. The discovery that selective chemical vapor deposition of iron from Fe(CO)(5) precursor on dielectric surfaces over tungsten surfaces is the key factor that enables the self-alignment of the iron nanowires. Dimensions of the wires are determined by the thickness of the thermal oxide. (c) 2007 The Electrochemical Society.

Attention Engagement in Early Infancy

We report a longitudinal study investigating developmental changes in the structure of attention engagement during early infancy. Forty-three infants were observed monthly from 2 to 4 months. Attention engagement was assessed from play interactions with parents, using a coding system developed by Bakeman and Adamson (1984). The results indicated a developmental transition in attention engagement at 3 months: after this age infants engaged for longer periods and in a wider variety of states. Most infants displayed person engagement at 2 months, passive joint engagement at 3 months, and object engagement at 4 months. To address whether emerging abilities of attention engagement allow infants to follow the attention of social partners, we compared attention engagement to performance on an experimental measure of attention control (reported by Perra & Gattis, 2010). Analyses revealed a positive relation between passive joint engagement and checking back, suggesting that changes in passive joint engagement reflect the development in attention control.

Selective Orthosteric Free Fatty Acid Receptor 2 (FFA2) Agonists IDENTIFICATION OF THE STRUCTURAL AND CHEMICAL REQUIREMENTS FOR SELECTIVE ACTIVATION OF FFA2 VERSUS FFA3

Free fatty acid receptor 2 (FFA2; GPR43) is a G protein-coupled seven-transmembrane receptor for short-chain fatty acids (SCFAs) that is implicated in inflammatory and metabolic disorders. The SCFA propionate has close to optimal ligand efficiency for FFA2 and can hence be considered as highly potent given its size. Propionate, however, does not discriminate between FFA2 and the closely related receptor FFA3 (GPR41). To identify FFA2-selective ligands and understand the molecular basis for FFA2 selectivity, a targeted library of small carboxylic acids was examined using holistic, label-free dynamic mass redistribution technology for primary screening and the receptor-proximal G protein [S-35] guanosine 5'-(3-O-thio) triphosphate activation, inositol phosphate, and cAMP accumulation assays for hit confirmation. Structure-activity relationship analysis allowed formulation of a general rule to predict selectivity for small carboxylic acids at the orthosteric binding site where ligands with substituted sp(3)-hybridized alpha-carbons preferentially activate FFA3, whereas ligands with sp(2)- or sp-hybridized alpha-carbons prefer FFA2. The orthosteric binding mode was verified by site-directed mutagenesis: replacement of orthosteric site arginine residues by alanine in FFA2 prevented ligand binding, and molecular modeling predicted the detailed mode of binding. Based on this, selective mutation of three residues to their non-conserved counterparts in FFA3 was sufficient to transfer FFA3 selectivity to FFA2. Thus, selective activation of FFA2 via the orthosteric site is achievable with rather small ligands, a finding with significant implications for the rational design of therapeutic compounds selectively targeting the SCFA receptors.

Spontaneous Attention to Faces in Asperger Sndrome using Ecologically Valid Static Stimuli

Previous eye tracking research on the allocation of attention to social information by individuals with autism spectrum disorders is equivocal and may be in part a consequence of variation in stimuli used between studies. The current study explored attention allocation to faces, and within faces,
by individuals with Asperger syndrome using a range of static stimuli where faces were either viewed in isolation or viewed in the context of a social scene. Results showed that faces were viewed typically by the individuals with Asperger syndrome when presented in isolation, but attention to the eyes was significantly diminished in comparison to age and IQ-matched typical viewers when faces were viewed as part of social scenes. We show that when using static stimuli, there is evidence of atypicality for individuals with Asperger syndrome depending on the extent of social context. Our findings shed light on the previous explanations of gaze behaviour that have emphasised the role of movement in atypicalities of social attention in autism spectrum disorders and highlight the importance of consideration of the realistic portrayal of social information for future studies.

Real-life Social Attention in Children with Autism Spectrum Disorders

Autism is a neuro-developmental disorder defined by atypical social behaviour, of which atypical social attention behaviours are among the earliest clinical markers (Volkmar et al., 1997). Eye tracking studies using still images and movie clips have provided a method for the precise quantification of atypical social attention in ASD. This is generally characterised by diminished viewing of the most socially pertinent regions (eyes), and increased viewing of less socially informative regions (body, background, objects) (Klin et al., 2002; Riby & Hancock, 2008, 2009). Ecological validity within eye tracking studies has become an increasingly important issue. As of yet, however, little is known about the precise nature of the atypicalities of social attention in ASD in real-life. Objectives: To capture and quantify gaze patterns for children with an ASD within a real life setting, compared to two Typically Developing (TD) comparison groups. Methods: Nine children with an ASD were compared to two age matched TD groups – a verbal (N=9) and a non-verbal (N=9) comparison group. A real-life scenario was created involving an experimenter posing as a magician, and consisted of 3 segments: a conversation segment; a magic trick segment; and a puppet segment. The first segment explored children’s attentional preferences during a real-life conversation; the magic trick segment explored children’s use of the eyes as a communicative cue, and the puppet segment explored attention capture. Finally, part of the puppet section explored children’s use of facial information in response to an unexpected event. Results: The most striking difference between the groups was the diminished viewing of the eyes by the ASD group in comparison to both control groups. This was found particularly during the conversation segment, but also during the magic trick segment, and during the puppet segment. When in conversation, participants with ASD were found to spend a greater proportion time looking off-screen, in comparison to TD participants. There was also a tendency for the ASD group to spend a greater proportion of time looking to the mouth of the experimenter. During the magic trick segment, despite the fact that the eyes were not predictive of a correct location, both TD comparison groups continued to use the eyes as a communicative cue, whereas the ASD group did not. In the puppet segment, all three groups spent a similar amount of time looking between the puppet and regions of the experimenter’s face. However, in response to an unexpected event, the ASD group were significantly slower to fixate back on the experimenter’s face. Conclusions: The results demonstrate the reduced salience of socially pertinent information for children with ASD in real life, and they provide support for the findings from previous eye tracking studies involving scene viewing. However, the results also highlight a pattern looking off-screen for both the TD and ASD groups. This eye movement behaviour is likely to be associated specifically with real-life interaction, as it has functional relevance (Doherty-Sneddon et al., 2002). However, the fact that it is significantly increased in the ASD group has implications for their understanding of real life social interactions.

Selective methane bromination over sulfated zirconia in SBA-15 catalysts

Methane activation via bromination can be a feasible route with selective synthesis of mono-bromomethane. It is known that the condensation of brominated products into higher hydrocarbons can result in coking and deactivation in the presence of di-bromomethane. In this study, selective production of methyl bromide was investigated over sulfated ZrO2 included SBA-15 structures. It was observed that the higher the ZrO2 amounts the higher the conversion, while the catalyst remained >99% selective for the monobrominated methane. Over 25 mol.% ZrO2 included SBA-15 catalyst with a BET surface area of 246 m(2)/g, methane was brominated with 69% conversion at 340 degrees C and only CH3Br was selectively produced. (C) 2009 Elsevier B.V. All rights reserved

Selective Hydrogenation of alpha,beta-Unsaturated Aldehydes and Ketones using Novel Manganese Oxide and Platinum Supported on Manganese Oxide Octahedral Molecular Sieves as Catalysts

The selective hydrogenation of ,-unsaturated aldehydes and ketones has been studied using ketoisophorone and cinnamaldehyde as model substrates using manganese oxide octahedral molecular sieve (OMS-2) based catalysts. For the first time, OMS-2 has been shown to be an efficient and selective hydrogenation catalyst. High selectivities for either the CC or CO double bond at approximate to 100% conversion were achieved by using OMS-2 and platinum supported on OMS-2 catalysts. Density functional theory (DFT) calculations showed that the dissociation of H2 on OMS-2 was water assisted and occurred on the surface Mn of OMS-2(001) that had been modified by an adsorbed H2O molecule. The theoretically calculated activation barrier was in good agreement with the experimentally determined value for the hydrogenation reactions, indicating that H2 dissociation on OMS-2 is likely to be the rate-determining step. A significant increase in the rate of reaction was observed in the presence of Pt as a result of the enhancement of H2 dissociative adsorption and subsequent reaction on the Pt or spillover of the hydrogen to the OMS-2 support. The relative adsorption strengths of ketoisophorone and cinnamaldehyde on the OMS-2 support compared with the Pt were found to determine the product selectivity.

An enantio-selective chromatographic stationary phase for S-ibuprofen prepared by stoichiometric molecular imprinting

Molecularly Imprinted Polymers (MIPs) against S-ibuprofen were synthesised using a tailor made functional monomer, 2-acrylamido-4-methylpyridine, following extensive pre-polymerisation studies of template-monomer complexation. An apparent association constant of 340 +/- 22 M-1 was calculated that was subsequently corrected to account for dimerisation of ibuprofen (K-dim = 320 +/- 95 M-1) resulting in an intrinsic association constant of 715 +/- 16 M-1, consistent with previously reported values. Using the synthesised imprinted polymer as a stationary phase, complete resolution of a racemic mixture of ibuprofen was achieved in predominantly aqueous mobile phases. An imprinting factor of 10 was observed, and was found to be in agreement with the difference in the average number of binding sites between MIP and blank polymers, calculated by staircase frontal chromatography. The imprinted polymers exhibited enhanced selectivity for the templated drug over structurally related NSAIDs. When applied as sorbents in solid-phase extraction of ibuprofen from commercial tablets, urine and blood serum samples, recoveries up to 92.2% were achieved. © The Royal Society of Chemistry 2012

Water-compatible imprinted polymers for selective depletion of riboflavine from beverages

Artificial riboflavin receptors adapted to aqueous environments were studied for their ability to selectively extract riboflavine (Rf) from three types of beverages i.e. milk, beer and a multivitamin mixture. The basic receptor was first prepared by molecular imprinting in nonaqueous medium using a hydrogen-bond donor-acceptor-donor functional monomer (2,6-bis(acrylamido)pyridine), complementary to the imide motif of the template, riboflavin tetra-acetate as template and pentaerythritol triacrylate (PETA) as a hydrophilic cross-linking monomer. The polymer was then packed in columns and used for extraction of riboflavine from beverages. Riboflavine (Rf) was selectively removed from milk and an artificial vitamin mixture but the nonspecific binding was still significant, as judged from the binding of Rf to a control nonimprinted polymer. In order to suppress this nonspecific binding, attempts to hydrolytically hydrophilize the polymer matrix were performed. The preferred approach consisted in a controlled base hydrolysis of pendent unreacted acrylate groups, using hydroxides with differently sized counterions as reagents. This resulted in a decreased binding of Rf to both polymers, but to an equal extent implying a preferential suppression of the nonspecific contribution to the binding. The hydrophilized polymers, when subjected to beer, showed larger imprinting factors at lower phase ratios compared to the nontreated polymers and a maximum removal of 86% compared to 47% for the nonimprinted control polymer.

Synthesis of caffeic acid and p-hydroxybenzoic acid molecularly imprinted polymers and their application for the selective extraction of polyphenols from olive mill waste waters

Using caffeic acid and p-hydroxybenzoic acid as templates, two molecularly imprinted polymers (MIPs) were prepared that were used for isolation of polyphenols from olive mill waste water samples (OMWWs) without previous pre-treatment. For the preparation of the caffeic acid MIPs 4-vinylpyridine, allylurea, allylaniline and methacrylic acid were tested as functional monomers, ethylene glycol dimethylacrylate (EDMA), pentaerythritol trimethylacrylate (PETRA) and divinylbenzene 80 (DVB80) as cross-linkers and tetrahydrofuran as porogen. For p-hydroxybenzoic acid 4-vinylpyridine, allylurea and allylaniline were tested as functional monomers, EDMA and PETRA as cross-linkers and acetonitrile as porogen. The performance of the synthesized polymers was evaluated against seven structurally related compounds by means of polymer-based HPLC. The two polymers that presented the most interesting properties were further evaluated by batch rebinding and from the derived isotherms their capacity and binding strength were determined. Using solid-phase extraction (SPE), their ability to recognize and bind the template molecule from an aqueous solution as well as the pH dependence of the binding strength were explored. After establishing the best SPE protocol, an aqueous model mixture of compounds and a raw OMWWs sample were loaded on the two best polymers. The result of the consecutive use of the two polymers on the same sample was explored. It was concluded that acidic conditions favour the recognition abilities of both polymers and that they can be used for a quick and efficient isolation of the polyphenol fraction directly from raw OMWW.

Selective solid-phase extraction sorbent for caffeine made by molecular imprinting

A molecularly imprinted polymer (MIP) was prepared with caffeine as the template molecule. Thermal polymerisation (60°C) was optimised, varying ratios of monomer, cross linker and template. The polymer was used as a solid-phase extraction (SPE) sorbent, for selective trapping and pre-concentration of caffeine. Caffeine was loaded on the MIP-SPE cartridge using different loading conditions (solvents, pH value). Washing and elution of the caffeine bound to the MIP was studied utilising different protocols. The extraction protocol was successfully applied to the direct extraction of caffeine from beverages and spiked human plasma.