The CRACAM Robot: Two-Dimensional Crystallization of Membrane Protein.

Membrane proteins are key cellular components that perform essential functions. They are major therapeutic targets. Electron crystallography can provide structural experimental information at atomic scale for membrane proteins forming two-dimensional (2D) crystals. There are two different methods to produce 2D crystals of membrane proteins. (1) either directly in the bulk of the solution (2) or under a lipid monolayer at the air-water interface. This extra lipid monolayer helps to pre-orient the proteins in order to facilitate the growth of 2D crystals. We present here these two methods for 2D crystallization of membrane proteins implemented in a fully automated robot called CRACAM. These methods require small volume of low concentration of proteins, making it possible to explore more conditions with the same amount of protein. These automated methods outperform traditional 2D crystallization approaches in terms of accuracy, flexibility, and throughput.

High energy resolution five-crystal spectrometer for high quality fluorescence and absorption measurements on an x-ray absorption spectroscopy beamline.

Fluorescence detection is classically achieved with a solid state detector (SSD) on x-ray absorption spectroscopy (XAS) beamlines. This kind of detection however presents some limitations related to the limited energy resolution and saturation. Crystal analyzer spectrometers (CAS) based on a Johann-type geometry have been developed to overcome these limitations. We have tested and installed such a system on the BM30B/CRG-FAME XAS beamline at the ESRF dedicated to the structural investigation of very dilute systems in environmental, material and biological sciences. The spectrometer has been designed to be a mobile device for easy integration in multi-purpose hard x-ray synchrotron beamlines or even with a laboratory x-ray source. The CAS allows to collect x-ray photons from a large solid angle with five spherically bent crystals. It will cover a large energy range allowing to probe fluorescence lines characteristic of all the elements from Ca (Z = 20) to U (Z = 92). It provides an energy resolution of 1-2 eV. XAS spectroscopy is the main application of this device even if other spectroscopic techniques (RIXS, XES, XRS, etc.) can be also achieved with it. The performances of the CAS are illustrated by two experiments that are difficult or impossible to perform with SSD and the complementarity of the CAS vs SSD detectors is discussed.

High-resolution spectroscopy on an X-ray absorption beamline.

A bent-crystal spectrometer based on the Rowland circle geometry has been installed and tested on the BM30b/FAME beamline at the European Synchrotron Radiation Facility to improve its performances. The energy resolution of the spectrometer allows different kinds of measurements to be performed, including X-ray absorption spectroscopy, resonant inelastic X-ray scattering and X-ray Raman scattering experiments. The simplicity of the experimental device makes it easily implemented on a classical X-ray absorption beamline. This improvement in the fluorescence detection is of particular importance when the probed element is embedded in a complex and/or heavy matrix, for example in environmental sciences.

Spherically bent analyzers for resonant inelastic X-ray scattering with intrinsic resolution below 200 meV.

Resonant inelastic X-ray scattering with very high energy resolution is a promising technique for investigating the electronic structure of strongly correlated materials. The demands for this technique are analyzers which deliver an energy resolution of the order of 200 meV full width at half-maximum or below, at energies corresponding to the K-edges of transition metals (Cu, Ni, Co etc.). To date, high resolution under these conditions has been achieved only with diced Ge analyzers working at the Cu K-edge. Here, by perfecting each aspect of the fabrication, it is shown that spherically bent Si analyzers can provide the required energy resolution. Such analyzers have been successfully produced and have greatly improved the energy resolution in standard spherically bent analyzers.

Bovine Nodular Thelitis: a Clinicopathological Study of 20 Cases.

Abstract- Bovine nodular thelitis in dairy cows is a chronic and enzootic infection. Four stages were distinguished: early stage, evolutive stage, ulcerative stage and final stage. Lesions were located mainly in the lower part of the udder or on the teat and recognized as solid papules and nodules. The histopathological study revealed a tuberculoid granulomatous pattern characterized by a subacute focal dermatitis with lymphoid cells, macrophages, epitheloid cells and a variable number of multinucleate giant cells (Langhans type) but no necrosis. Acid-fast bacilli were demonstrated in two of 14 cases. Three strains of acid fast bacilli were isolated but the only one identified was Mycobacterium terrae. M. terrae is a non-pathogenic slowly growing, non-photochromogenic mycobacterium. Its role in tuberculosis-like skin lesion is the bovine udder is uncertain. Résumé- La thélite nodulaire est une infection chronique et enzootique chez les vaches laitières. On distingue 4 phases: une phase précoce, une phase évolutive, une phase ulcérative et une phase finale. Vingt échantillons histologiques, mammelles et trayons, ont été examinés. Les lésions étaient localisées essentiellement à la partie basse de la mammelle ou sur le trayon et se présentaient sous l'aspect de papules solides ou de nodules. Les échantillons étaient divisés en deux parties: l'une destinée à l'isolement de mycobactéries, l'autre à l'examen histologique. Les lésions histologiques des trayons étaient des granulomes tuberculoïdes, caractérisés par une dermatite subaigue focale avec présence de lymphocytes, macrophages, cellules épithélioïdes et une nombre variable de cellules multinucléées géantes (type Langhans). Les infiltrats inflammatoires étaient principalement périvasculaires. Parfois, ils infiltraient toute la paroi du trayon. Aucune nécrose n'a été observée. Des bacilles acidoalcoolorésistants furent mis en évidence de manière inconstante (2 cas sur 14). Trois types de bacilles acidoalcoolorésistants ont été isolés raais, un seul a été identifié comme étant Mycobacterium terrae. M. terrae est une mycobactérie non pathogène, de croissance lente et non photochromogène. L'implication de cet organisme dans les lésions de type tuberculeux dans la mammelle de la vache est discutée. Zusammenfassung- Bei Milchkühen stellt die bovine noduläre Thelitis eine chronische und enzootische Infektion dar. Vier Stadien lassen sich unterscheiden: Früstadium evolutorisches Stadium, ulzeratives Stadium und Endstadium. 20 pathologische Proben von Euter und Zitzen wurden untersucht. Die Veränderungen waren hauptsächlich am unteren Teil des Euters oder an den Zitzen lokalisiert und ließen sich als solide Papeln oder Knoten ansprechen. Das Untersuchungsgul wurde für die Isolierung von Mykobakterien und für eine histopathologische Untersuchung geteilt. Die histopathologischen Veränderungen der Zitzen zeigten ein tuberkuloides granulomatöses Bild, das durch subakute fokale Dermatitis mit lymphyoiden Zellen, Makrophagen, Epitheloidzellen und einer variablen Anzahl von vielkernigen Riesenzellen (Langhans-Typ) gekennzeichnet war. Die entzündlichen Infiltrate befanden sich hauptsächlich perivaskulär. Manchmal ließen sie sich aber auch in der gesamten Zitzenwand nachweisen. Nekrosen bestaznden nicht. Säurefeste Bazillus-Keime waren inkonstant vorhanden (2 von 14 Fällen). Drei Arten von säurefesten Bazillus-Keimen wurden isoliert, aber nur eine wurde speziell identifiziert (Mycobacterium terrae). M. terrae ist ein nicht pathogenes, langsam wachsendes und nich photochromogenes Mykobakterium. Die Beteiligung dieses Organismus bei den tuberkulose-ähnlichen Hautveränderungen am bovinen Euter wird diskutiert. Resumen La Telitis Nodular Bovina es una infección enzoótica crónica de las vacas lecheras. Se distinguen cuatro etapas: etapa inicial, etapa evolutiva, etapa ulcerativa y etapa final. En el presente trabajo se examinaron 20 muestras de mama y de pezón. Las lesiones se localizaban principalmente en la parte inferior de la glándula mamaria o en el pezón y consistían en pápulas sólidas y en nódulos. Las muestras se dividían en dos partes, una para el aislamiento de micobacterias y la otra para los estudios histopatológicos. Las lesiones histopatológicas consistían en una reacción granulomatosa de tipo tuberculoide, caracterizada por una dermatitis focal subaguda con células linfoides, macrófagos, células epitelioides y un número variable de células gigantes multinucleadas (tipo Langhans). Los infiltrados inflamatorios estaban localizados predominantemente en la zone perivascular. En ocasiones, aparecían a lo largo de toda la pared de la glándula mamaria. No se observó necrosis. Se han aislado tres cepas de bacilos acido-alcohol resistentes, pero sólo una fue identificada específicamente como Mycobacterium terrae. M. Terrae es una micobacteria no fotocromogénica de crecimiento lento y no patógena. Se discute la posible participación de este microorganismo en la génesis de las lesiones pseudotuberculosas observadas en estos animales.