Cell Surface Display Yields Evolvable, Clickable Antibody Fragments

Non-canonical amino acids (ncAAs) provide powerful tools for engineering the chemical and physical properties of proteins. However, introducing ncAAs into proteins can affect protein properties in unpredictable ways, thus necessitating screening efforts to identify mutants with desirable properties. In this work, we describe an Escherichia coli cell surface display platform for the directed evolution of clickable antibody fragments. This platform enabled isolation of antibody fragments with improved digoxigenin binding and modest affinity maturation in several different ncAA contexts. Azide-functionalized fragments exhibited improved binding kinetics relative to their methionine counterparts, facile chemical modification through azide–alkyne cycloaddition, and retention of binding properties after modification. The results described here suggest new possibilities for protein engineering, including modulation of molecular recognition events by ncAAs and direct screening of libraries of chemically modified proteins

Overexpression of hepatic prothymosin alpha, a novel marker for human hepatocellular carcinoma.

Identification of gene products exclusively or abundantly expressed in cancer may yield novel tumour markers. We recently isolated a number of cDNA clones, including alpha-prothymosin, from rat hepatocellular carcinoma (HCC) using a subtraction-enhanced display technique. Alpha-Prothymosin is involved in cell proliferation and is regulated by the oncogene c-myc in vitro. In the present study, we analysed alpha-prothymosin gene expression and its correlation with c-myc in patients with HCC, cirrhosis and adenoma and in normal controls. Hepatic alpha-prothymosin messenger RNA (mRNA) levels were two- to 9.2-fold higher in tumoral tissues than in adjacent non-tumoral tissues in 14 of 17 patients with HCC, regardless of coexisting cirrhosis and viral hepatitis. No marked difference in alpha-prothymosin mRNA levels was present in patients with adenoma and hepatic cirrhosis and in healthy controls. The c-myc mRNA amounts were two- to fivefold increased in 11 of 17 patients with HCC and correlated significantly with those of alpha-prothymosin (P < 0.001). In situ hybridization revealed that increased alpha-prothymosin mRNA was localized in the tumour nodules of the patients with HCC. These data suggest that overexpression of alpha-prothymosin in HCC patients, correlated with c-myc, is possibly involved in the tumorigenic process and may be a novel molecular marker for human HCC

Visible-Light Photoswitchable Benzimidazole Azo-Arenes as beta-Arrestin2-Biased Selective Cannabinoid 2 Receptor Agonists

Acknowledgements The authors would like to acknowledge Dr. Andrea Holme for excellent technical support and the Iain Fraser Cytometry Centre (University of Aberdeen) for providing access to their equipment. The authors would like to thank Dr. Matthias Scheiner for his contributions towards the development of the calcium mobilization assay and Dr. Valérie Jahns for her efforts towards faster automated analysis of the obtained results. Nick Verhavert is acknowledged for his assistance with the NanoBiT® assay. Diego Rodriguez-Soacha is acknowledged for establishing the rCB1R radioligand binding assay in our laboratory. Special thanks to Dr. Rangan Maitra and RTI International for providing the G16 coupled hCB1 and hCB2 CHO-K1 cell lines. The authors thank Nadine Yurdagül-Hemmrich and Annette Hannawacker for excellent technical support. This project was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft under DFG DE1546/10-1). J. N. Hislop’s financing support was given by NHS Grampian. The research visit of S. A. M. Steinmüller in Dr. Hislop’s laboratory was funded by the Elite Network of Bavaria (grant N° K-BM-2013-247). J. Fender and A. Tutov were supported by the International Doctoral Program “Receptor Dynamics” funded within the framework of the Elite Network of Bavaria (grant N° K-BM-2013- 247). M. H. Deventer was funded by the Research FoundationFlanders (FWO; grant 1S54521N).Peer reviewe

The influence of rice husk ash addition on the properties of metakaolin-based geopolymers

This paper investigates the replacement of metakaolin (MK) with rice husk ash (RHA) in the production of alkali-activated binders or geopolymers. The influence of the RHA addition on compressive and flexural strength, as well as water absorption and apparent porosity were determined, in terms of the percentage of RHA in the mixture and molar ratios of the mixes. Fourier Transform Infrared (FTIR) spectroscopy and Energy Dispersive spectroscopy (EDS) were carried out to assess the changes in the microstructure of the geopolymer matrices with the RHA addition. Results have shown that RHA may be a supplementary precursor for geopolymers. The composition of the geopolymer matrices containing 0-40% RHA is very similar, which indicates that the additional Si provided by RHA is not incorporated to the geopolymer matrix. In addition, geopolymers with RHA content higher than 40% present a plastic behavior, characterized by extremely low strength and high deformation, which can be attributed to the formation of silica gel in formulations containing variable Si/Al ratio

Alkaline activation of ceramic waste materials

Ceramic materials represent around 45 % of construction and demolition waste, and originate not only from the building process, but also as rejected bricks and tiles from industry. Despite the fact that these wastes are mostly used as road sub-base or construction backfill materials, they can also be employed as supplementary cementitious materials, or even as raw material for alkali-activated binders This research aimed to investigate the properties and microstructure of alkali-activated cement pastes and mortars produced from ceramic waste materials of various origins. Sodium hydroxide and sodium silicate were used to prepare the activating solution. The compressive strength of the developed mortars ranged between 22 and 41 MPa after 7 days of curing at 65 C, depending on the sodium concentration in the solution and the water/binder ratio. These results demonstrate the possibility of using alkaliactivated ceramic materials in building applications.The authors are grateful to the Spanish Ministry of Science and Innovation for supporting this study through Project GEOCEDEM BIA 2011-26947, and also to FEDER funding. They also thank Universitat Jaume I for supporting this research through Lucia Reig's granted research stay.Reig Cerdá, L.; Mitsuuchi Tashima, M.; Soriano, L.; Borrachero Rosado, MV.; Monzó Balbuena, JM.; Paya Bernabeu, JJ. (2013). Alkaline activation of ceramic waste materials. Waste and Biomass Valorization. 4:729-736. https://doi.org/10.1007/s12649-013-9197-zS7297364Puertas, F., García-Díaz, I., Barba, A., Gazulla, M.F., Palacios, M., Gómez, M.P., Martínez-Ramírez, S.: Ceramic wastes as alternative raw materials for Portland cement clinker production. Cement Concrete Comp. 30(9), 798–805 (2008)Ministerio de Fomento de España, Catálogo de Residuos Utilizables en Construcción (2010). http://www.cedexmateriales.vsf.es/view/catalogo.aspx . Retrieved on 6 Dec 2012Stock, D.: World production and consumption of ceramic tiles. Tile Today 73, 50–58 (2011)Medina, C., Juan, A., Frías, M., Sánchez-de-Rojas, M.I., Morán, J.M., Guerra, M.I.: Characterization of concrete made with recycled aggregate from ceramic sanitary ware. Mater. Construcc. 61(304), 533–546 (2011)Pacheco-Torgal, F., Jalali, S.: Reusing ceramic wastes in concrete. Constr. Build. Mater. 24(5), 832–838 (2010)Lavat, A.E., Trezza, M.A., Poggi, M.: Characterization of ceramic roof tile wastes as pozzolanic admixture. Waste Manage. 29(5), 1666–1674 (2009)Nuran, A., Mevlut, U.: The use of waste ceramic tile in cement production. Cement Concrete Res. 30, 497–499 (2000)Pereira-de-Oliveira, L.A., Castro-Gomes, J.P., Santos, P.M.S.: The potential pozzolanic activity of glass and red-clay ceramic waste as cement mortars components. Constr. Build. Mater. 31, 197–203 (2012)Van Deventer, J.S.J., Provis, J.L., Duxson, P., Brice, D.G.: Chemical research and climate change as drivers in the commercial adoption of alkali activated materials. Waste Biomass Valor. 1, 145–155 (2010)van Deventer, J.S.J., Provis, J.L., Duxson, P., Lukey, G.C.: Reaction mechanisms in the geopolymeric conversion of inorganic waste to useful products. J. Hazard. Mater. A139, 506–513 (2007)Duxson, P., Fernández-Jiménez, A., Provis, J.L., Lukey, G.C., Palomo, A., van Deventer, J.S.J.: Geopolymer technology: the current state of the art. J. Mater. Sci. 42(9), 2917–2993 (2007)Bernal, S.A., Rodríguez, E.D., de Gutiérrez, R.M., Provis, J.L., Delvasto, S.: Activation of metakaolin/slag blends using alkaline solutions based on chemically modified silica fume and rice husk ash. Waste Biomass Valor. 3, 99–108 (2012)Fernández-Jiménez, A., Palomo, A., Criado, M.: Microstructure development of alkali-activated fly ash cement: a descriptive model. Cement Concrete Res 35, 1204–1209 (2005)Payá, J., Borrachero, M.V., Monzó, J., Soriano, L., Tashima, M.M.: A new geopolymeric binder from hydrated-carbonated cement. Mater. Lett. 74, 223–225 (2012)Kourti, I., Amutha-Rani, D., Deegan, D., Boccaccini, A.R., Cheeseman, C.R.: Production of geopolymers using glass produced from DC plasma treatment of air pollution control (APC) residues. J. Hazard. Mater. 176, 704–709 (2010)Puertas, F., Barba, A., Gazulla, M.F., Gómez, M.P., Palacios, M., Martínez-Ramírez, S.: Residuos cerámicos para su posible uso como materia prima en la fabricación de clínker de cemento Portland: caracterización y activación alcalina. Mater. Construcc. 56(281), 73–84 (2006)Reig, L., Tashima, M.M., Borrachero, M.V., Monzó, J., Payá, J.: Nuevas matrices cementantes generadas por Activación Alcalina de residuos cerámicos. II Simposio Aprovechamiento de residuos agro-industriales como fuente sostenible de materiales de construcción, November 8–9, Valencia, Spain, pp. 199–207 (2010)L. Reig, M.M. Tashima, M.V. Borrachero, J. Monzó, J. Payá: Residuos de ladrillos cerámicos en la producción de conglomerantes activados alcalinamente, I Pro-Africa Conference: Non-conventional Building Materials Based on Agroindustrial Wastes, October 18–19, Pirassununga, SP, Brazil, pp. 18–21 (2010)García Ten F.J. Descomposición durante la cocción del carbonato cálcico contenido en el soporte crudo de los azulejos. Tesis de doctorado, Departamento de Ingeniería química, UJI (2005)Baronio, G., Binda, L.: Study of the pozzolanicity of some bricks and clays. Constr. Build. Mater. 11(1), 41–46 (1997)Zanelli, C., Raimondo, M., Guarini, G., Dondi, M.: The vitreous phase of porcelain stoneware: composition, evolution during sintering and physical properties. J. Non-Cryst. Solids 357, 3251–3260 (2011)Carty, W.M., Senapati, U.: Porcelain-raw materials, processing, phase evolution, and mechanical behaviour. J. Am. Ceram. Soc. 81(1), 3–20 (1998)ASCER, COACV, COPUT, ITC-AICE, WEBER ET BROUTIN – CEMARKSA: Guía Baldosa Guía de la baldosa cerámica. IVE: Conselleria d’Obres Públiques, Urbanisme i Transports, 4ª Ed. Valencia (2003)Khater, H.M.: Effect of calcium on geopolimerization of aluminosilicate wastes. J. Mater. Civ. Eng. 24, 92–101 (2012)Bondar, D., Lynsdale, C.J., Milestone, N.B., Hassani, N., Ramezanianpour, A.A.: Effect of adding mineral additives to alkali-activated natural pozzolan paste. Constr. Build. Mater. 25, 2906–2910 (2011)Provis, J.L., Harrex, R.M., Bernal, A.S., Duxson, P., van Deventer, J.S.J.: Dilatometry of geopolymers as a means of selecting desirable fly ash sources. J. Non-Cryst. Solids 358, 1930–1937 (2012)Duxson, P., Provis, J.L., Lukey, G.C., Mallicoat, S.W., Kriven, W.M., van Deventer, J.S.J.: Understanding the relationship between geopolymer composition, microstructure and mechanical properties. Colloid Surf. A 269, 47–58 (2005)Tashima, M.M., Akasaki, J.L., Castaldelli, V.N., Soriano, L., Monzó, J., Payá, J., Borrachero, M.V.: New geopolymeric binder based on fluid catalytic cracking catalyst residue (FCC). Mater. Lett. 80, 50–52 (2012)Komnitsas, K., Zaharaki, D., Perdikatsis, V.: Geopolymerisation of low calcium ferronickel slags. J. Mater. Sci. 42, 3073–3082 (2007)Bernal, S.A., Gutierrez, R.M., Provis, J.L., Rose, V.: Effect of silicate modulus and metakaolin incorporation on the carbonation of alkali silicate-activated slags. Cement Concrete Res. 40, 898–907 (2010)Tashima, M.M. Produccion y caracterizacion de materiales cementantes a partir del silicoaluminato calcico vitreo (VCAS). Tesis de doctorado, Departamento de Ingeniería de la construcción y de proyectos de ingeniería civil, UPV (2012)Provis, J.L., van Deventer, J.S.J.: Geopolymerisation kinetics. 2. Reaction kinetic modelling. Chem. Eng. Sci. 62, 2318–2329 (2007

Hypercoagulability progresses to hypocoagulability during evolution of acetaminophen-induced acute liver injury in pigs

Increases in prothrombin time (PT) and international normalised ratio (INR) characterise acute liver injury (ALI) and failure (ALF), yet a wide heterogeneity in clotting abnormalities exists. This study defines evolution of coagulopathy in 10 pigs with acetaminophen (APAP)-induced ALI compared to 3 Controls. APAP administration began at 0 h and continued to ‘ALF’, defined as INR >3. In APAP pigs, INR was 1.05 ± 0.02 at 0 h, 2.15 ± 0.43 at 16 h and > 3 at 18 ± 1 h. At 12 h thromboelastography (TEG) demonstrated increased clot formation rate, associated with portal vein platelet aggregates and reductions in protein C, protein S, antithrombin and A Disintegrin and Metalloprotease with Thrombospondin type 1 repeats–13 (ADAMTS-13) to 60%, 24%, 47% and 32% normal respectively. At 18 ± 1 h, INR > 3 was associated with: hypocoagulable TEG profile with heparin-like effect; falls in thrombin generation, Factor V and Factor VIII to 52%, 19% and 17% normal respectively; further decline in anticoagulants; thrombocytopenia; neutrophilia and endotoxemia. Multivariate analysis, found that ADAMTS-13 was an independent predictor of a hypercoagulable TEG profile and platelet count, endotoxin, Protein C and fibrinogen were independent predictors of a hypocoagulable TEG profile. INR remained normal in Controls. Dynamic changes in coagulation occur with progression of ALI: a pro-thrombotic state progresses to hypocoagulability

Dual-Acting Small Molecules: Subtype-Selective Cannabinoid Receptor 2 Agonist/Butyrylcholinesterase Inhibitor Hybrids Show Neuroprotection in an Alzheimer's Disease Mouse Model.

We present the synthesis and characterization of merged human butyrylcholinesterase (hBChE) inhibitor/cannabinoid receptor 2 (hCB2R) ligands for the treatment of neurodegeneration. In total, 15 benzimidazole carbamates were synthesized and tested for their inhibition of human cholinesterases, also with regard to their pseudoirreversible binding mode and affinity toward both cannabinoid receptors in radioligand binding studies. After evaluation in a calcium mobilization assay as well as a β-arrestin 2 (βarr2) recruitment assay, two compounds with balanced activities on both targets were tested for their immunomodulatory effect on microglia activation and regarding their pharmacokinetic properties and blood-brain barrier penetration. Compound 15d, containing a dimethyl carbamate motif, was further evaluated in vivo, showing prevention of Aβ25-35-induced learning impairments in a pharmacological mouse model of Alzheimer's disease for both short- and long-term memory responses. Additional combination studies proved a synergic effect of BChE inhibition and CB2R activation in vivo

COSNET-a coherent optical subscriber network

A complete coherent multichannel system, designed for application in the local loop, is presented. The concept of a uni- and bidirectional system and its technical realization in a laboratory demonstrator are described. The network control, including frequency management of the bidirectional channels, and network security are discussed. Attention is paid to the scenario for evolution from a narrowband to a complete broadband system. All aspects are integrated in a demonstrator, which is capable of supporting a large number of narrowband and broadband distributive and communicative services. Novel technical solutions for frequency management, data induced polarization switching (DIPS), high-speed encryption, and network signaling are presented

Obituary J. W. ten Cate

Thrombosis and Hemostasi