Microinjection of specific anti-IMPDH2 antibodies induces disassembly of cytoplasmic rods/rings that are primarily stationary and stable structures

Background: Our laboratory previously reported interesting rods 3-10 mu m long and rings 2-5 mu m diameter (RR) in the cytoplasm of mammalian cells. Experimental evidence show that both inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) and cytidine triphosphate synthetase (CTPS) are components of RR structures. Several cell types, including mouse embryonic stem cells, and cell lines, such as mouse 3 T3 and rat NRK, naturally present RR structures, while other cells can present RR when treated with compounds interfering with GTP/CTP biosynthetic pathways. in this study, we aimed to investigate the dynamic behavior of these RR in live cells.Results: RR were detected in > 90% of COS-7 and HeLa cells treated with 1 mM ribavirin or 6-Diazo-5-oxo-L-norleucine (DON) for 24 h, and in 75% of COS-7 cells treated with 1 mM mycophenolic acid (MPA) for the same period of time. Microinjection of affinity-purified anti-IMPDH2 antibodies in live COS-7 cells treated with ribavirin, DON, or MPA showed mature forms of RR presented as stable and stationary structures in 71% of cells. in the remaining 29% of cells, RR acquired erratic movement and progressively disassembled into fragments and disappeared within 10 min. the specific stationary state and antibody-dependent disassembling of RR structures was independently confirmed in COS-7 and HeLa cells transfected with GFP-tagged IMPDH2.Conclusions: This is the first demonstration of disassembly of RR structures upon microinjection of anti-IMPDH2 antibodies that led to the disappearance of the molecular aggregates. the disassembly of RR after microinjection of anti-IMPDH2 antibody further strengthens the notion that IMPDH2 are major building blocks of RR. Using two independent methods, this study demonstrated that the induced RR are primarily stationary structures in live cells and that IMPDH2 is a key component of RR.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ Florida, Dept Oral Biol, Gainesville, FL 32610 USAUniversidade Federal de São Paulo, Div Rheumatol, BR-04023062 São Paulo, BrazilFleury Med & Hlth Labs, Div Immunol, BR-04102050 São Paulo, BrazilUniv Idaho, Dept Biol Sci, Moscow, ID 83844 USAUniversidade Federal de São Paulo, Div Rheumatol, BR-04023062 São Paulo, BrazilCAPES: 9028-11-0FAPESP: 2011/12448-0Web of Scienc

Comet assay analysis of single–stranded DNA breaks in circulating leukocytes of glaucoma patients

PURPOSE: To investigate the amount of single-stranded DNA breaks in circulating leukocytes of primary open-angle glaucoma (POAG) patients. METHODS: A comparative quantification of DNA breaks was performed in circulating leukocytes of POAG patients and healthy controls. The following groups of subjects were compared: (1) POAG patients having primary vascular dysregulation (PVD), (2) POAG patients without PVD, (3) healthy controls with PVD, and (4) healthy controls without PVD. The damage to DNA resulting in single-stranded breaks was assessed by means of the alkaline comet assay in which the damaged DNA migrates out of the nucleus forming a tail, which can be quantified using image analysis. Damage was quantified as the comet tail moment, which represents the extent of DNA damage in individual cells. RESULTS: Leukocytes of POAG patients exerted a significantly higher amount of comet tails, which are indicative of DNA damage, in comparison to control leukocytes (p>0.001). DNA breaks occurred particularly in the subgroup of POAG patients with PVD in comparison to glaucoma patients without PVD (p=0.002). In the control group, there was no significant difference between controls with PVD and controls without PVD (p=0.86). CONCLUSIONS: POAG patients with PVD have a significantly higher rate of DNA breaks than both POAG patients without PVD and healthy controls with and without PVD

"Quasi two-dimensional" spin distributions in II-VI magnetic semiconductor heterostructures: Clustering and dimensionality

Spin clustering in diluted magnetic semiconductors (DMS) arises from antiferromagnetic exchange between neighboring magnetic cations and is a strong function of reduced dimensionality. Epitaxially-grown single monolayers and abrupt interfaces of DMS are, however, never perfectly two-dimensional (2D) due to the unavoidable inter-monolayer mixing of atoms during growth. Thus the magnetization of DMS heterostructures, which is strongly modified by spin clustering, is intermediate between that of 2D and 3D spin distributions. We present an exact calculation of spin clustering applicable to arbitrary distributions of magnetic spins in the growth direction. The results reveal a surprising insensitivity of the magnetization to the form of the intermixing profile, and identify important limits on the maximum possible magnetization. High-field optical studies of heterostructures containing "quasi-2D" spin distributions are compared with calculation.Comment: 5 pages (RevTeX), 5 embedded EPS figs, published in PRB v61 p1736 (2000

Oscillating magnetoresistance in diluted magnetic semiconductor barrier structures

Ballistic spin polarized transport through diluted magnetic semiconductor (DMS) single and double barrier structures is investigated theoretically using a two-component model. The tunneling magnetoresistance (TMR) of the system exhibits oscillating behavior when the magnetic field are varied. An interesting beat pattern in the TMR and spin polarization is found for different NMS/DMS double barrier structures which arises from an interplay between the spin-up and spin-down electron channels which are splitted by the s-d exchange interaction.Comment: 4 pages, 6 figures, submitted to Phys. Rev.

Insights into the Mechanism of Ligand Binding to Octopine Dehydrogenase from Pecten maximus by NMR and Crystallography

Octopine dehydrogenase (OcDH) from the adductor muscle of the great scallop, Pecten maximus, catalyzes the NADH dependent, reductive condensation of L-arginine and pyruvate to octopine, NAD+, and water during escape swimming and/or subsequent recovery. The structure of OcDH was recently solved and a reaction mechanism was proposed which implied an ordered binding of NADH, L-arginine and finally pyruvate. Here, the order of substrate binding as well as the underlying conformational changes were investigated by NMR confirming the model derived from the crystal structures. Furthermore, the crystal structure of the OcDH/NADH/agmatine complex was determined which suggests a key role of the side chain of L-arginine in protein cataylsis. Thus, the order of substrate binding to OcDH as well as the molecular signals involved in octopine formation can now be described in molecular detail

Quantifying signal changes in nano-wire based biosensors

In this work, we present a computational methodology for predicting the change in signal (conductance sensitivity) of a nano-BioFET sensor (a sensor based on a biomolecule binding another biomolecule attached to a nano-wire field effect transistor) upon binding its target molecule. The methodology is a combination of the screening model of surface charge sensors in liquids developed by Brandbyge and co-workers [Sørensen et al., Appl. Phys. Lett., 2007, 91, 102105], with the PROPKA method for predicting the pH-dependent charge of proteins and protein-ligand complexes, developed by Jensen and co-workers [Li et al., Proteins: Struct., Funct., Bioinf., 2005, 61, 704-721, Bas et al., Proteins: Struct., Funct., Bioinf., 2008, 73, 765-783]. The predicted change in conductance sensitivity based on this methodology is compared to previously published data on nano-BioFET sensors obtained by other groups. In addition, the conductance sensitivity dependence from various parameters is explored for a standard wire, representative of a typical experimental setup. In general, the experimental data can be reproduced with sufficient accuracy to help interpret them. The method has the potential for even more quantitative predictions when key experimental parameters (such as the charge carrier density of the nano-wire or receptor density on the device surface) can be determined (and reported) more accurately. © 2011 The Royal Society of Chemistry

Finding a voice: A figured worlds approach to theorising young children's identities

This article explores some of the ways in which children’s ethnic identities have been conceptualised by sociocultural and critical race theory and the potential of the ‘figured worlds’ literature in helping to theorise the responses of young children to the cultural and educational worlds they encounter. Using some vignettes drawn from the author’s ethnographic study of the ethnic identities of a group of 3- and 4-year-old White British and British Pakistani children in a kindergarten in the north of England, the article explores the potential of a figured worlds analysis in understanding how the children respond to some of the experiences of the kindergarten and in understanding how they seek to make sense of their identities. The article concludes that while structural and cultural factors shaped the ways in which the children engaged or did not engage in the social and educational practices of the kindergarten and played a very significant part in how they viewed themselves and viewed others, the children were not silent observers of what the world offered or did not offer them. A dialogic self was evident that authored and tried to make sense of the world, but, in so doing, designated identities meant that only particular figured worlds were available to children for much of the time. It is argued that what a figured worlds reading offers is a means of seeking to uncover and theorise the complex ways in which young children experience and perform their identities and respond to the social and educational practices in particular contexts. This is seen as having value in providing a framework for early childhood academics and educators to work together to support children in exploring alternative figured identities that challenge, alleviate and transform the constraints that positional identities often seem to impose on them

“After Lunch We Offer Quiet Time and Meditation” : Early Learning Environments in Australia and Finland Through the Lenses of Educators

Modern societies organize ECEC services from their own cultural, social and political contexts, which is also reflected in the steering documents of the country and further in the work of teachers (Garvis, et al., 2018). In many of the countries children’s access to preschool has broadened and the benefits of high quality ECEC have been recognized. In Australia and Finland, concepts of play based learning, child initiated play or free play have been highlighted as founding pillars of the early learning environments. In this paper we take a closer look at ECEC environments in Australia and Finland through the lenses of 26 educators. They described in an online questionnaire children’s daily activities as well as they indicated the amount of free play related to these activities.Peer reviewe

The electrophotonic silicon biosensor

The emergence of personalized and stratified medicine requires label-free and low-cost diagnostic technology capable of monitoring multiple disease biomarkers in parallel. Silicon photonic biosensors combine high sensitivity analysis with scalable, low-cost manufacturing technology but they tend to measure only a single biomarker and provide no information about their (bio)chemical activity. Here, we introduce an electrochemical silicon photonic sensor capable of highly sensitive and multiparameter profiling of biomolecules. Our electro-photonic technology consists of microring resonators optimally n-doped to support high Q resonances alongside electrochemical processes in situ. The inclusion of electrochemical processes enables site selective immobilization of different biomolecules, here single stranded DNA, onto individual microrings within a sensor array. The combination of photonic and electrochemical characterization of molecules bound to the sensor surface also provides direct quantification of binding density and unique insight into chemical reactivity that is unavailable with photonic detection alone. By exploiting both the photonic and the electrical properties of silicon, the sensor opens new modalities for sensing on the micro-scale

Highly selective BSA imprinted polyacrylamide hydrogels facilitated by a metal-coding MIP approach

We report the fabrication of metal-coded molecularly imprinted polymers (MIPs) using hydrogel-based protein imprinting techniques. A Co(II) complex was prepared using (E)-2-((2 hydrazide-(4-vinylbenzyl) hydrazono)methyl)phenol; along with iron(III) chloroprotoporphyrin (Hemin), vinylferrocene (VFc), zinc (II) protoporphyrin (ZnPP) and protoporphyrin (PP), these complexes were introduced into the MIPs as co-monomers for metal-coding of non-metalloprotein imprints. Results indicate a 66% enhancement for bovine serum albumin (BSA) protein binding capacities (Q, mg/g) via metal-ion/ligand exchange properties within the metal-coded MIPs. Specifically, Co(II)-complex-based MIPs exhibited 92 ± 1% specific binding with Q values of 5.7 ± 0.45 mg BSA/g polymer and imprinting factors (IF) of 14.8 ± 1.9 (MIP/non-imprinted (NIP) control). The selectivity of our Co(II)-coded BSA MIPs were also tested using bovine haemoglobin (BHb), lysozyme (Lyz), and trypsin (Tryp). By evaluating imprinting factors (K), each of the latter proteins was found to have lower affinities in comparison to cognate BSA template. The hydrogels were further characterised by thermal analysis and differential scanning calorimetry (DSC) to assess optimum polymer composition