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Purpose The purpose of this study was to elucidate the role and molecular consequences of impaired glutathione (GSH) biosynthesis on eye development. Methods GSH biosynthesis was impaired in surface ectoderm-derived ocular tissues by crossing Gclcf/f mice with hemizygous Le-Cre transgenic mice to produce Gclcf/f/Le-CreTg/- (KO) mice. Control mice included Gclcf/f and Gclcwt/wt/Le-CreTg/- mice (CRE). Eyes from all mice (at various stages of eye development) were subjected to histological, immunohistochemical, Western blot, RT-qPCR, RNA-seq, and subsequent Gene Ontology, Ingenuity Pathway Analysis and TRANSFAC analyses. PAX6 transactivation activity was studied using a luciferase reporter assay in HEK293T cells depleted of GSH using buthionine sulfoximine (BSO). Results Deletion of Gclc diminished GSH levels, increased reactive oxygen species (ROS), and caused an overt microphthalmia phenotype characterized by malformation of the cornea, iris, lens, and retina that is distinct from and much more profound than the one observed in CRE mice. In addition, only the lenses of KO mice displayed reduced crystallin (α, β), PITX3 and Foxe3 expression. RNA-seq analyses at postnatal day 1 revealed 1552 differentially expressed genes (DEGs) in the lenses of KO mice relative to those from Gclcf/f mice, with Crystallin and lens fiber cell identity genes being downregulated while lens epithelial cell identity and immune response genes were upregulated. Bioinformatic analysis of the DEGs implicated PAX6 as a key upstream regulator. PAX6 transactivation activity was impaired in BSO-treated HEK293T cells. Conclusions These data suggest that impaired ocular GSH biosynthesis may disrupt eye development and PAX6 function.

Recent reports have challenged the notion that the lens is immune-privileged. However, these studies have not fully identified the molecular mechanism(s) that promote immune surveillance of the lens. Using a mouse model of targeted glutathione (GSH) deficiency in ocular surface tissues, we have investigated the role of oxidative stress in upregulating cytokine expression and promoting immune surveillance of the eye. RNA-sequencing of lenses from postnatal day (P) 1-aged Gclc(f/f);Le-Cre(Tg/-) (KO) and Gclc(f/f);Le-Cre(−/−) control (CON) mice revealed upregulation of many cytokines (e.g., CCL4, GDF15, CSF1) and immune response genes in the lenses of KO mice. The eyes of KO mice had a greater number of cells in the aqueous and vitreous humors at P1, P20 and P50 than age-matched CON and Gclc(w/w);Le-Cre(Tg/-) (CRE) mice. Histological analyses revealed the presence of innate immune cells (i.e., macrophages, leukocytes) in ocular structures of the KO mice. At P20, the expression of cytokines and ROS content was higher in the lenses of KO mice than in those from age-matched CRE and CON mice, suggesting that oxidative stress may induce cytokine expression. In vitro administration of the oxidant, hydrogen peroxide, and the depletion of GSH (using buthionine sulfoximine (BSO)) in 21EM15 lens epithelial cells induced cytokine expression, an effect that was prevented by co-treatment of the cells with N-acetyl-L-cysteine (NAC), a antioxidant. The in vivo and ex vivo induction of cytokine expression by oxidative stress was associated with the expression of markers of epithelial-to-mesenchymal transition (EMT), α-SMA, in lens cells. Given that EMT of lens epithelial cells causes posterior capsule opacification (PCO), we propose that oxidative stress induces cytokine expression, EMT and the development of PCO in a positive feedback loop. Collectively these data indicate that oxidative stress induces inflammation of lens cells which promotes immune surveillance of ocular structures.

Samarium-doped ZrB2/SiC (ZBS) coatings possess properties of high emissivity and excellent ablation performance suitable for hypersonic applications. Of interest in the current study is how cyclic ablation affects the scale development on alumina substrates. ZBS coatings with 3, 5 and 8 mol% of samarium (Sm) dopant were prepared via shrouded plasma spray onto alumina substrates and subjected to two 60-s ablation cycles with temperatures reaching up to 1700 °C. Blisters were observed on the Sm-doped coatings after the 1st cycle as a result of a local eutectic reaction between the ablation products and alumina substrate. A Sm-stabilized t-ZrO2 phase was identified through X-ray diffraction after the ablation of the Sm-doped coatings. The ZBS with 5 mol% of Sm dopant produced a flower-like microstructure after the 2nd cycle due to the formation of convection cells.

The temperature of leading edges during hypersonic flight can be reduced by maximizing the emissivity of their surface. In this study, the concentration of a known emissivity modifier (Sm) has been varied (3, 5, and 8 mol% Sm in ZrB 2 /SiC coatings) and the total hemispherical emissivity and ablation resistance of the coatings has been evaluated. Maximum emissivity was observed to occur for the 5 mol% Sm ZrB 2 /SiC coating, with a value of 0.9 measured at 1600 °C. All the ablated Sm-doped coatings have an oxide scale consisting of m-ZrO 2 and c 1 -Sm 0.2 Zr 0.9 O 1.9, and/or Sm 2 Zr 2 O 7 depending on the Sm concentration. Large amounts of porosity were observed in the oxide scale of the ablated 8 mol% Sm ZrB 2 /SiC coating. This was attributed to viscosity reduction of the B 2 O 3 and SiO 2 glassy phases by the Sm 3+ and their subsequent vaporization. The 5 mol% Sm-doped ZrB 2 /SiC demonstrated the optimal emissivity properties and dense oxide scale formation.

Aromatic compounds represent a broad class of chemicals with a range of industrial applications, all of which are conventionally derived from petroleum feedstocks. However, owing to a diversity of available pathway precursors along with natural and engineered enzyme 'parts', microbial cell factories can be engineered to create alternative, renewable routes to many of the same aromatic products. Drawing from the latest tools and strategies in metabolic engineering and synthetic biology, such efforts are becoming an increasingly systematic practice, while continued efforts promise to open new doors to an ever-expanding range and diversity of renewable chemical and material products. This short review will highlight recent and notable achievements related for the microbial production of aromatic chemicals.

p -Hydroxybenzoate, protocatechuate, and catechol represent fine and/or commodity chemicals useful as antioxidants and building-block molecules. To date, however, these species have been largely overlooked as focal end-products. An existing route employing protocatechuate and catechol as intermediates suffers from the need for multiple auxotrophies to preserve precursor (3-dehydroshikimate) availability. A novel, modular route from endogenous p -hydroxybenzoate has been engineered in Escherichia coli for the individual biosynthesis of all three products from renewable glucose while minimizing auxotrophy generation. To enhance endogenous biosynthesis of p -hydroxybenzoate, native chorismate pyruvate lyase ( ubiC ) was over-expressed. p -Hydroxybenzoate was converted to protocatechuate by a hydroxylase ( pobA ) from Pseudomonas aeruginosa . Catechol was produced by the additional co-expression of protocatechuate decarboxylase from Enterobacter cloacae . Systematic expression of appropriate pathway elements in phenylalanine overproducing E. coli enabled initial titers of 32 ± 4, 110 ± 8, and 81 ± 15 mg/L for p -hydroxybenzoate, protocatechuate, and catechol, respectively. Disruption of chorismate mutase/prephenate dehydratase ( pheA ) to preserve endogenous chorismate then allowed maximum titers of 277 ± 2, 454 ± 11, and 451 ± 44 mg/L, respectively, at glucose yields of 5.8, 9.7, and 14.3% of their respective theoretical maxima. Catechol titers were further improved to 630 ± 37 mg/L in a batch bioreactor study. The proposed pathway can furthermore serve as a platform for other bioproducts, including the bioplastics precursor cis,cis -muconate.

A variety of bibliometric measures have been proposed to quantify the impact of researchers and their work. The h-index is a notable and widely-used example which aims to improve over simple metrics such as raw counts of papers or citations. However, a limitation of this measure is that it considers authors in isolation and does not account for contributions through a collaborative team. To address this, we propose a natural variant that we dub the Social h-index. The idea is to redistribute the h-index score to reflect an individual's impact on the research community. In addition to describing this new measure, we provide examples, discuss its properties, and contrast with other measures., Comment: 5 pages, 3 figures, 1 table

ObjectivesThe goal of this study was to develop and validate a new fibrin-targeted imaging agent that enables high-resolution near-infrared fluorescence (NIRF) imaging of deep vein thrombosis (DVT).BackgroundNIRF imaging of fibrin could enable highly sensitive and noninvasive molecular imaging of thrombosis syndromes in vivo.MethodsA fibrin-targeted peptide was conjugated to a near-infrared fluorophore Cy7, termed FTP11-Cy7. The NIRF peptide is based on a fibrin-specific imaging agent that has completed Phase II clinical magnetic resonance imaging trials. In vitro binding of FTP11-Cy7 to human plasma clots was assessed by using fluorescence reflectance imaging. Next, FTP11-Cy7 was intravenously injected in mice with femoral DVT induced by topical 7.5% ferric chloride treatment. Intravital fluorescence microscopy and noninvasive fluorescence molecular tomography–computed tomography were performed in 32 mice with DVT, followed by histological analyses.ResultsIn vitro human clot-binding analyses showed a 6-fold higher NIRF clot target-to-background ratio (TBR) of FTP11-Cy7 than free Cy7 (6.3 ± 0.34 vs. 1.2 ± 0.03; p < 0.0001). The thrombus TBR of acute and subacute femoral DVT with FTP11-Cy7 obtained by using intravital fluorescence microscopy was >400% higher than control free Cy7. Binding of FTP11-Cy7 to thrombi was blocked by a 100-fold excess of unlabeled competitor peptide both in vitro and in vivo (p < 0.001 for each). Histological analyses confirmed that FTP11-Cy7 specifically accumulated in thrombi. Noninvasive fluorescence molecular tomography–computed tomography imaging of fibrin in jugular DVT demonstrated strong NIRF signal in thrombi compared with sham-operated jugular veins (mean TBR 3.5 ± 0.7 vs. 1.5 ± 0.3; p < 0.05).ConclusionsThe fibrin-targeted NIRF agent FTP11-Cy7 was shown to avidly and specifically bind human and murine thrombi, and enable sensitive, multimodal intravital and noninvasive NIRF molecular imaging detection of acute and subacute murine DVT in vivo.

G protein-coupled receptors (GPCRs) convey extracellular stimulation into dynamic intracellular action, leading to the regulation of cell migration and differentiation. T lymphocytes express G alpha(i2) and G alpha(i3), two members of the G alpha(i/o) protein family, but whether these two G alpha(i) proteins have distinguishable roles guiding T cell migration remains largely unknown because of a lack of member-specific inhibitors. This study details distinct G alpha(i2) and G alpha(i3) effects on chemokine receptor CXCR3-mediated signaling. Our data showed that G alpha(i2) was indispensable for T cell responses to three CXCR3 ligands, CXCL9, CXCL10, and CXCL11, as the lack of G alpha(i2) abolished CXCR3-stimulated migration and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) incorporation. In sharp contrast, T cells isolated from G alpha(i3) knock-out mice displayed a significant increase in both GTPgammaS incorporation and migration as compared with wild type T cells when stimulated with CXCR3 agonists. The increased GTPgammaS incorporation was blocked by G alpha(i3) protein in a dose-dependent manner. G alpha(i3)-mediated blockade of G alpha(i2) activation did not result from G alpha(i3) activation, but instead resulted from competition or steric hindrance of G alpha(i2) interaction with the CXCR3 receptor via the N terminus of the second intracellular loop. A mutation in this domain abrogated not only G alpha(i2) activation induced by a CXCR3 agonist but also the interaction of G alpha(i3) to the CXCR3 receptor. These findings reveal for the first time an interplay of G alpha(i) proteins in transmitting G protein-coupled receptor signals. This interplay has heretofore been masked by the use of pertussis toxin, a broad inhibitor of the G alpha(i/o) protein family.

Tests are made of an aspect of the "knowledge sources" theoretical account of acquisition of reading in which, contrary to the developmental bypass hypothesis, it is postulated that sublexical relations between orthographic and phonological components are formed very early in learning by spontaneous induction from stored print word experience. Experiments 1 and 2, conducted with 5- and 6-year-old children, indicated as predicted that positional frequency of orthographic components in experienced print words influenced reading responses to unfamiliar pseudoword items. In Experiment 3 positional frequency of an orthographic component was manipulated in a training-transfer paradigm. Transfer to pseudoword reading was as predicted. The results could not be given alternative explanations by the developmental bypass hypothesis nor by accounts which predict exclusive use of onset and rime units at this early reading level.

This Report provides a package of reform proposals for tribunals in Northern Ireland. There are two elements to the project. The first is a small, qualitative study of users of three different tribunals to understand the users' experience of tribunals in Northern Ireland. The second element is a scoping study which explored, in interviews, the parameters, operational issues, possibilities and difficulties of tribunal reform, as well as the current gap in the arrangements for the oversight and accountability of tribunals. The Report's recommendations are presented around the themes of users, processes, judicial leadership, structures, system review and a roadmap indicating tasks and a schedule for their implementation.

B570 Centrosomal Clustering: A Novel Therapeutic Target for Multiple Myeloma MS Raab, I Breitkreutz, B Rebacz, PJ Hayden, TO Larsen, MH Clausen, JH Fruehauf, H Goldschmidt, KC Anderson, A Kraemer Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; CCU Molecular Hematology/ Oncology, German Cancer Research Center, Heidelberg, Germany; BioCentrum, Technical University of Denmark, Kopenhagen, Denmark; Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; National Center of Tumor Diseases, Dept. of Medicine, University of Heidelberg, Germany

Then focused on the RTK’s which were shown to be upregulated in WM and tested the effect of TKI-258 effect on their activity. Moreover, we tested the effect of TKI-258 survival, proliferation, and apoptosis of WM cells, and its effect of different signaling pathways in WM. Results: We found that epidermal growth factor receptor (EGF-R), ephrin receptor (Eph-R) and receptor tyrosine kinase–like orphan receptor 1 (ROR-1) were highly activated in WM patients compared to normal subjects (5, 10, and 14-fold increase, respectively), and were found to be upregulated also in BCWM1. TKI-258 reduced the activity of EGF-R and Eph-R, but not ROR1. Moreover, TKI-258 induced cytotoxicity, prevented proliferation and induced apoptosis in WM cells in a dose dependent manner with IC50 of 500-800 nM. Moreover, it was shown to induce cleavage of PARP, caspase-3 and caspase-9, and also reduced phosphorylations of ERK1/2. Conclusion: We identified two novel therapeutic targets for WM; EGF-R and Eph-R, and identified Eph-R as a novel target of TKI-258. Moreover, TKI-258 reduced WM progression in vitro, and we are now testing the effect of TKI-258 on WM progression in vitro and alone and in combination with other drugs.