Polystyrene beads as an alternative support material for epitope identification of a prion-antibody interaction using proteolytic excision-mass spectrometry

The binding epitope structure of a protein specifically recognized by an antibody provides key information to prevent and treat diseases with therapeutic antibodies and to develop antibody-based diagnostics. Epitope structures of antigens can be effectively identified by the proteolytic epitope excision-mass spectrometry (MS) method, which involves (1) immobilization of monoclonal or polyclonal antibodies, e.g., on N-hydroxysuccinimide-activated sepharose, (2) affinity binding of the antigen followed by limited proteolytic digestion of the immobilized immune complex, and (3) elution and mass spectrometric analysis of the remaining affinity-bound peptide(s). In the epitope analysis of recombinant cellular bovine prion protein (bPrPC) to a monoclonal antibody (mAb3E7), we found that epitope excision experiments resulted in extensive nonspecific binding of bPrP to a standard sepharose matrix employed. Here, we show that the use of amino-modified polystyrene beads with aldehyde functionality is an efficient alternative support for antibody immobilization, suitable for epitope excision-MS, with complete suppression of nonspecific bPrP bindin

Lectin-sugar interactions deciphered by SPR-MS and Credrex-MS

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Growth of organic crystals via attachment and transformation of nanoscopic precursors

A key requirement for the understanding of crystal growth is to detect how new layers form and grow at the nanoscale. Multistage crystallization pathways involving liquid-like, amorphous or metastable crystalline precursors have been predicted by theoretical work and have been observed experimentally. Nevertheless, there is no clear evidence that any of these precursors can also be relevant for the growth of crystals of organic compounds. Herein, we present a new growth mode for crystals of DL-glutamic acid monohydrate that proceeds through the attachment of preformed nanoscopic species from solution, their subsequent decrease in height at the surface and final transformation into crystalline 2D nuclei that eventually build new molecular layers by further monomer incorporation. This alternative mechanism provides a direct proof for the existence of multistage pathways in the crystallization of molecular compounds and the relevance of precursor units larger than the monomeric constituents in the actual stage of growth.publishe

N-Glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry

Intercellular adhesion molecule-1 (ICAM-1) is a heavily N‐glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and ∼4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2‐inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylate

Large Scale Optimization Problems for Central Energy Facilities with Distributed Energy Storage

On large campuses, energy facilities are used to serve the heating and cooling needs of all the buildings, while utilizing cost savings strategies to manage operational cost. Strategies range from shifting loads to participating in utility programs that offer payouts. Among available strategies are central plant optimization, electrical energy storage, participation in utility demand response programs, and manipulating the temperature setpoints in the campus buildings. However, simultaneously optimizing all of the central plant assets, temperature setpoints and participation in utility programs can be a daunting task even for a powerful computer if the desire is real time control. These strategies may be implemented separately across several optimization systems without a coordinating algorithm. Due to system interactions, decentralized control may be far from optimal and worse yet may try to use the same asset for different goals. In this work, a hierarchal optimization system has been created to coordinate the optimization of the central plant, the battery, participation in demand response programs, and temperature setpoints. In the hierarchal controller, the high level coordinator determines the load allocations across the campus or facility. The coordinator also determines the participation in utility incentive programs. It is shown that these incentive programs can be grouped into reservation programs and price adjustment programs. The second tier of control is split into 3 portions: control of the central energy facility, control of the battery system, and control of the temperature setpoints. The second tier is responsible for converting load allocations into central plant temperature setpoints and flows, battery charge and discharge setpoints, and temperature setpoints, which are delivered to the Building Automation System for execution. It is shown that the whole system can be coordinated by representing the second tier controllers with a smaller set of data that can be used by the coordinating controller. The central plant optimizer must supply an operational domain which constrains how each group of equipment can operate. The high level controller uses this information to send down loadings for each resource a group of equipment in the plant produces or consumes. For battery storage, the coordinating controller uses a simple integrator model of the battery and is responsible for providing a demand target and the amount of participation in any incentive programs. Finally, to perform temperature setpoint optimization a dynamic model of the zone is provided to the coordinating controller. This information is used to determine load allocations for groups of zones. The hierarchal control strategy is successful at optimizing the entire energy facility fast enough to allow the algorithms to control the energy facility, building setpoints, and program bids in real-time

“A very orderly retreat”: Democratic transition in East Germany, 1989-90

East Germany's 1989-90 democratisation is among the best known of East European transitions, but does not lend itself to comparative analysis, due to the singular way in which political reform and democratic consolidation were subsumed by Germany's unification process. Yet aspects of East Germany's democratisation have proved amenable to comparative approaches. This article reviews the comparative literature that refers to East Germany, and finds a schism between those who designate East Germany's transition “regime collapse” and others who contend that it exemplifies “transition through extrication”. It inquires into the merits of each position and finds in favour of the latter. Drawing on primary and secondary literature, as well as archival and interview sources, it portrays a communist elite that was, to a large extent, prepared to adapt to changing circumstances and capable of learning from “reference states” such as Poland. Although East Germany was the Soviet state in which the positions of existing elites were most threatened by democratic transition, here too a surprising number succeeded in maintaining their position while filing across the bridge to market society. A concluding section outlines the alchemy through which their bureaucratic power was transmuted into property and influence in the “new Germany”

Latimerija - živi fosil

This study, which derives from work initiated as part of a N.A.T.O. Collaborative Linkage Grant (LST‐CLG No. 979499), supported by consecutive research contracts (SF0238 and SF0248) from the UK Department of Environment, Food and Rural Affairs (to GHC), which were complemented by a Marie Curie postdoctoral fellowship (PIEF‐GA‐2008‐219707) and a subsequent research grant from the National Science Centre, Poland (decision No DEC‐2011/01/D/NZ8/01807) to GZ.Carotenoid‐based pigmentation is a striking feature of many taxa, yet the function, if any, of colour traits is often unclear. Pumpkinseed Lepomis gibbosus, a widely introduced freshwater sunfish that exhibits alternative male mating strategies, express a striking, red operculum spot. To investigate the potential function of this red spot as a signal in this species' mating system, we determined the presence and measured the size of red operculum spots in fish collected from 12 populations in five European countries in which pumpkinseed is an established non‐native species. We subsequently related the presence and size of the red spot to body size and mating strategy, based on an analysis of relative gonad size (gonado‐somatic index, GSI), using a mixed modelling approach. The study demonstrated that the presence of a red operculum spot in pumpkinseed is associated with sexual maturation, with GSI frequency distributions suggesting that cuckolders in some non‐native populations comprised both sneaker and satellite males, the latter not having previously been reported for this species. Further, the size of red spot correlated strongly with body size in parental and cuckolder males, although there was no difference in the presence or size of the red operculum spot between male mating strategies. The function of a red operculum spot in females is not clear but may be partly mediated by pleiotropic genetic mechanisms. Red operculum spots appear to function as signals of male maturation and body size in pumpkinseed, irrespective of mating strategy.PostprintPeer reviewe

The AIDS and Cancer Specimen Resource: Role in HIV/AIDS scientific discovery

The AIDS Cancer and Specimen Resource (ACSR) supports scientific discovery in the area of HIV/AIDS-associated malignancies. The ACSR was established as a cooperative agreement between the NCI (Office of the Director, Division of Cancer Treatment and Diagnosis) and regional consortia, University of California, San Francisco (West Coast), George Washington University (East Coast) and Ohio State University (Mid-Region) to collect, preserve and disperse HIV-related tissues and biologic fluids and controls along with clinical data to qualified investigators. The available biological samples with clinical data and the application process are described on the ACSR web site. The ACSR tissue bank has more than 100,000 human HIV positive specimens that represent different processing (43), specimen (15), and anatomical site (50) types. The ACSR provides special biospecimen collections and prepares speciality items, e.g., tissue microarrays (TMA), DNA libraries. Requests have been greatest for Kaposi's sarcoma (32%) and non-Hodgkin's lymphoma (26%). Dispersed requests include 83% tissue (frozen and paraffin embedded), 18% plasma/serum and 9% other. ACSR also provides tissue microarrays of, e.g., Kaposi's sarcoma and non-Hodgkin's lymphoma, for biomarker assays and has developed collaborations with other groups that provide access to additional AIDS-related malignancy specimens. ACSR members and associates have completed 63 podium and poster presentations. Investigators have submitted 125 letters of intent requests. Discoveries using ACSR have been reported in 61 scientific publications in notable journals with an average impact factor of 7. The ACSR promotes the scientific exploration of the relationship between HIV/AIDS and malignancy by participation at national and international scientific meetings, contact with investigators who have productive research in this area and identifying, collecting, preserving, enhancing, and dispersing HIV/AIDS-related malignancy specimens to funded, approved researchers at no fee. Scientific discovery has been advanced by this unique biorepository. Investigators are encouraged to browse the ACSR Internet site for materials to enhance their own scientific initiatives