The generation and characterisation of mice with conditional knock-out of the NMDA receptor subunit NR2B

The N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors. They play a critical role in neuronal pattern formation during development and in synaptic plasticity as molecular coincidence detectors. NMDA receptor is a tetrameric protein complex comprised of two obligatory NR1 subunits and two identical or different NR2 subunits, of which four types exist named NR2A-D. In rodents and other mammals, NR1 and NR2B are expressed in the entire central nervous system, already at embryonic stages, whereas NR2A expression starts and increases only postnatally to coexist with NR2B in the adult brain. Mice lacking the NR1 subunit or lacking the NR2B subunit die at birth, whereas mice lacking NR2A are viable. Both NR2A and NR2B containing NMDA receptors are implicated in synaptic plasticity, learning and memory formation but their distinctive functions are unknown. The NR2B subunit received a lot of attention because mice genetically altered to overexpress NR2B showed improved spatial reference memory and enhanced LTP. The lethality of the general NR2B knock-out gives rise to the necessity of a conditional knock-out, by which deleterious effects due to lack of NR2B during embryogenesis are prevented, and the physiological function of NR2B can be elucidated in the postnatal brain. For this purpose, a DNA construct for homologous recombination in embryonic stem (ES) cells was generated with NR2B allele exon 6 flanked by loxP sequences. This exon encodes the region preceding the first transmembrane domain of the NR2B subunit. As a selection marker, a neomycin resistance gene flanked by frt sites was introduced in intron 6. The selection marker was subsequently removed by flp recombinase from the targeted NR2B allele, and the ES cells were used for blastocyst injection to derive NR2B2lox mice. NR2B2lox mice were bred with TgCre4 mice, selectively expressing Cre recombinase in forebrain principal neurons, to generate mice heterozygous for the transgene TgCre4 and homozygous for the conditional NR2B allele (NR2B2lox/2lox). In these mice (NR2B(delta)Fb), postnatal forebrain principal neurons should lack NMDA receptors containing the NR2B subtype. Deletion of NR2B in NR2B(delta)Fb mice was revealed by electrophysiological measurements. In parallel, in this study, also lentiviral mediated gene delivery was used in vivo for Cre/loxP mediated DNA recombination. Recombinant lentivirus encoding Cre recombinase and the GFP protein under the control of the alpha-CaMKII promoter was delivered stereotactically to the hippocampal CA1 region of NR2B2lox/2lox mice at P20. Lack of NR2B was assessed by electrophysiological measurements of synaptic and whole-cell NMDA currents, using NR2B specific antagonists. Recordings from CA1 neurons revealed reduced NMDA currents, lack of sensitivity to ifenprodil, a selective blocker of NR2B containing NMDA receptors, and faster than wild type deactivation kinetics of NMDA mediated currents, indicating the effective loss of the NR2B-type NMDA receptors. Frequency and AMPA component of miniature EPSCs were unaltered whereas the NMDA component was reduced. Moreover an impairment of cellular LTP could be shown. Western blot analysis from hippocampal homogenates of NR2B(delta)Fb mice showed a 70% reduction of the NR2B subunit levels, and no significant change in the expression levels of NR2A, and of the AMPA receptor subunits GluR-A and GluR-B. Collectively, these studies describe a conditional mouse model for elucidating the particular physiological functions of the NR2B type of NMDA receptors in the adult forebrain

Combined XPS and contact angle studies of ethylene vinyl acetate and polyvinyl acetate blends

Cataloged from PDF version of article.In this study, we prepared thin films by blending ethylene vinyl acetate copolymers (EVA) containing 12-33 (wt.%) vinyl acetate (VA) with polyvinyl acetate (PVAc) and high density polyethylene homopolymers. Large area micropatterns having controlled protrusion sizes were obtained by phase-separation especially for the PVAc/EVA-33 blends using dip coating. These surfaces were characterized by XPS and contact angle measurements. A reasonably linear relation was found between the VA content on the surface (wt.%) obtained from XPS analysis and the VA content in bulk especially for PVAc/EVA-33 blend surfaces. PE segments were more enriched on the surface than that of the bulk for pure EVA copolymer surfaces similar to previous reports and VA enrichment was found on the EVA/HDPE blend surfaces due to high molecular weight of HDPE. Water theta(e) decreased with the increase in the VA content on the blend surface due to the polarity of VA. A good agreement was obtained between gamma(-)(s) and atomic oxygen surface concentration with the increase of VA content. The applicability of Cassie-Baxter equation was tested and found that it gave consistent results with the experimental water contact angles for the case where VA content was lower than 55 wt.% in the bulk composition. (C) 2011 Elsevier B. V. All rights reserved

A probable outbreak of toxoplasmosis among boarding school students in Turkey

ABSTRACTIn total, 171 students from a boarding school in Izmir, Turkey, with mild and non-specific symptoms of toxoplasmosis, were screened during September–October 2002. All 171 students were seropositive for Toxoplasma gondii IgG and IgM. Of 43 students tested, 40 (93%) had low IgG avidity. None showed evidence of ophthalmic involvement. The data suggest that T. gondii may spread rapidly in close living conditions, possibly following exposure to cat litter. This is the largest recent outbreak of toxoplamosis described in the medical literature

Effectiveness Of Continuous Versus Pulsed Short-Wave Diathermy In The Management Of Knee Osteoarthritis: A Randomised Pilot Study

Background: Short-wave diathermy (SWD) is an electrotherapeutic modality used in the conservative treatment of knee osteoarthritis (KOA).  Electromagnetic radiation delivered in continuous (cSWD) or pulse (pSWD) mode provides a deep heating effect on tissues.  There is no consensus on outcomes of treatment with cSWD versus pSWD in KOA. The aim of this study was to compare the effects of cSWD versus pSWD on pain, functionality and walking distance in KOA. Methods: 34 female patients aged 49-65 with KOA were randomized into two groups.  A total of 27 patients completed the study. One group (n=11) was treated with cSWD, the other (n=16) with pSWD for three weeks. Patients were assessed before, after and at one month post therapy. Outcome measures included visual analogue scale (VAS) for knee pain, Western Ontario and Mcmaster University Osteoarthritis Index (WOMAC) and a six-minute walking test (6MWT). Results: Based on the minimal clinically important improvement (MCII), there was a reduction in VAS and WOMAC scores in both cSWD and pSWD groups post treatment (-37.3mm, 31.2mm respectively for VAS and 26, 23 respectively for WOMAC) and at one month post treatment. There was no difference in pre and post treatment VAS for pain, WOMAC or 6MWT scores between the two groups.  There was a small post treatment effect size on between- group 6MWT scores (Cohen’s d: 0.238). Conclusion: Both treatment options appear to be efficacious in reducing pain and improving functionality in KOA.  There was no between-group difference. A larger study must be conducted to consolidate these findings.   &#160

Highly Fluorescent Pyrene-Functional Polystyrene Copolymer Nanofibers for Enhanced Sensing Performance of TNT

A pyrene-functional polystyrene copolymer was prepared via 1,3-dipolar cycloaddition reaction (Sharpless-type click recation) between azide-functional styrene copolymer and 1-ethynylpyrene. Subsequently, nanofibers of pyrene-functional polystyrene copolymer were obtained by using electrospinning technique. The nanofibers thus obtained, found to preserve their parent fluorescence nature, confirmed the avoidance of aggregation during fiber formation. The trace detection of trinitrotoluene (TNT) in water with a detection limit of 5 nM was demonstrated, which is much lower than the maximum allowable limit set by the U.S. Environmental Protection Agency. Interestingly, the sensing performance was found to be selective toward TNT in water, even in the presence of higher concentrations of toxic metal pollutants such as Cd2+, Co2+, Cu2+, and Hg2+. The enhanced sensing performance was found to be due to the enlarged contact area and intrinsic nanoporous fiber morphology. Effortlessly, the visual colorimetric sensing performance can be seen by naked eye with a color change in a response time of few seconds. Furthermore, vapor-phase detection of TNT was studied, and the results are discussed herein. In terms of practical application, electrospun nanofibrous web of pyrene-functional polystyrene copolymer has various salient features including flexibility, reproducibility, and ease of use, and visual outputs increase their value and add to their advantage. © 2015 American Chemical Society

Characterisation of the Immunophenotype of Dogs with Primary Immune-Mediated Haemolytic Anaemia

Immune-mediated haemolytic anaemia (IMHA) is reported to be the most common autoimmune disease of dogs, resulting in significant morbidity and mortality in affected animals. Haemolysis is caused by the action of autoantibodies, but the immunological changes that result in their production have not been elucidated.To investigate the frequency of regulatory T cells (Tregs) and other lymphocyte subsets and to measure serum concentrations of cytokines and peripheral blood mononuclear cell expression of cytokine genes in dogs with IMHA, healthy dogs and dogs with inflammatory diseases.19 dogs with primary IMHA, 22 dogs with inflammatory diseases and 32 healthy control dogs.Residual EDTA-anti-coagulated blood samples were stained with fluorophore-conjugated monoclonal antibodies and analysed by flow cytometry to identify Tregs and other lymphocyte subsets. Total RNA was also extracted from peripheral blood mononuclear cells to investigate cytokine gene expression, and concentrations of serum cytokines (interleukins 2, 6 10, CXCL-8 and tumour necrosis factor α) were measured using enhanced chemiluminescent assays. Principal component analysis was used to investigate latent variables that might explain variability in the entire dataset.There was no difference in the frequency or absolute numbers of Tregs among groups, nor in the proportions of other lymphocyte subsets. The concentrations of pro-inflammatory cytokines were greater in dogs with IMHA compared to healthy controls, but the concentration of IL-10 and the expression of cytokine genes did not differ between groups. Principal component analysis identified four components that explained the majority of the variability in the dataset, which seemed to correspond to different aspects of the immune response.The immunophenotype of dogs with IMHA differed from that of dogs with inflammatory diseases and from healthy control dogs; some of these changes could suggest abnormalities in peripheral tolerance that permit development of autoimmune disease. The frequency of Tregs did not differ between groups, suggesting that deficiency in the number of these cells is not responsible for development of IMHA