Electron Microscopy Reveals Structural and Chemical Changes at the Nanometer Scale in the Osteogenesis Imperfecta Murine Pathology

Alternations of collagen and mineral at the molecular level may have a significant impact on the strength and toughness of bone. In this study, scanning transmission electron microscopy (STEM) and electron energy-loss spectroscopy (EELS) were employed to study structural and compositional changes in bone pathology at nanometer spatial resolution. Tail tendon and femoral bone of osteogenesis imperfecta murine (oim, brittle bone disease) and wild type (WT) mice were compared to reveal defects in the architecture and chemistry of the collagen and collagen-mineral composite in the oim tissue at the molecular level. There were marked differences in the substructure and organization of the collagen fibrils in the oim tail tendon; some regions have clear fibril banding and organization, while in other regions fibrils are disorganized. Malformed collagen fibrils were loosely packed, often bent and devoid of banding pattern. In bone, differences were detected in the chemical composition of mineral in oim and WT. While mineral present in WT and oim bone exhibited the major characteristics of apatite, examination in EELS of the fine structure of the carbon K ionization edge revealed a significant variation in the presence of carbonate in different regions of bone. Variations have been also observed in the fine structure and peak intensities of the nitrogen K-edge. These alterations are suggestive of differences in the maturation of collagen nucleation sites or cross-links. Future studies will aim to establish the scale and impact of the modifications observed in oim tissues. The compositional and structural alterations at the molecular level cause deficiencies at larger length scales. Understanding the effect of molecular alterations to pathologic bone is critical to the design of effective therapeutics

Semantic-based policy engineering for autonomic systems

This paper presents some important directions in the use of ontology-based semantics in achieving the vision of Autonomic Communications. We examine the requirements of Autonomic Communication with a focus on the demanding needs of ubiquitous computing environments, with an emphasis on the requirements shared with Autonomic Computing. We observe that ontologies provide a strong mechanism for addressing the heterogeneity in user task requirements, managed resources, services and context. We then present two complimentary approaches that exploit ontology-based knowledge in support of autonomic communications: service-oriented models for policy engineering and dynamic semantic queries using content-based networks. The paper concludes with a discussion of the major research challenges such approaches raise

Rad51 Paralogs Remodel Pre-synaptic Rad51 Filaments to Stimulate Homologous Recombination

SummaryRepair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which nucleates RAD-51-ssDNA filaments, RFS-1/RIP-1 binds and remodels pre-synaptic filaments to a stabilized, “open,” and flexible conformation, in which the ssDNA is more accessible to nuclease digestion and RAD-51 dissociation rate is reduced. Walker box mutations in RFS-1, which abolish filament remodeling, fail to stimulate RAD-51 strand exchange activity, demonstrating that remodeling is essential for RFS-1/RIP-1 function. We propose that Rad51 paralogs stimulate HR by remodeling the Rad51 filament, priming it for strand exchange with the template duplex

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy

The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocytederived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research

Active Coordination in Ad Hoc Networks

Abstract. The increasing ubiquity of communicating mobile devices and vastly different mobile application needs have led to the emergence of middleware models for ad hoc networks that simplify application pro-gramming. One such system, EgoSpaces, addresses specific needs of indi-vidual applications, allowing them to define what data is included in their operating context using declarative specifications constraining properties of data, agents that own the data, hosts on which those agents are run-ning, and attributes of the ad hoc network. In the resulting coordination model, application agents interact with a dynamically changing environ-ment through a set of views, or custom defined projections of the set of data present in the surrounding ad hoc network. This paper builds on EgoSpaces by allowing agents to assign behaviors to their personal-ized views. Behaviors consist of actions that are automatically performed in response to specified changes in a view. Behaviors discussed in this paper encompass reactive programming, transparent data migration, au-tomatic data duplication, and event capture. Formal semantic definitions and programming examples are given for each behavior.

Preparation of Sterically Demanding 2,2-Disubstituted-2-Hydroxy Acids by Enzymatic Hydrolysis

Preparation of optically-pure derivatives of 2-hydroxy-2-(3-hydroxyphenyl)-2-phenylacetic acid of general structure 2 was accomplished by enzymatic hydrolysis of the correspondent esters. A screening with commercial hydrolases using the methyl ester of 2-hydroxy-2-(3-hydroxyphenyl)-2-phenylacetic acid (1a) showed that crude pig liver esterase (PLE) was the only preparation with catalytic activity. Low enantioselectivity was observed with substrates 1a\u2013d, whereas PLE-catalysed hydrolysis of 1e proceeded with good enantioselectivity (E = 28), after optimization. Enhancement of the enantioselectivity was obtained by chemical re-esterification of enantiomerically enriched 2e, followed by sequential enzymatic hydrolysis with PLE. The preparation of optically-pure (S)-2e was validated on multi-milligram scale

A facility for radiation hardness studies based on a medical cyclotron

The development of instrumentation for operation in high-radiation environments represents a challenge in various research fields, particularly in particle physics experiments and space missions, and drives an ever-increasing demand for irradiation facilities dedicated to radiation hardness studies. Depending on the application, different needs arise in terms of particle type, energy and dose rate. In this article, we present a versatile installation based on a medical cyclotron located at the Bern University Hospital (Inselspital), which is used as a controlled 18-MeV proton source. This accelerator is used for daily production of medical radioisotopes, as well as for multidisciplinary research, thanks to a 6.5-meter long beam transfer line that terminates in an independent bunker, dedicated only to scientific activities. The facility offers a wide range of proton fluxes, due to an adjustable beam current from approximately 10 pA to the micro-ampere range, together with a series of steering and focusing magnets along the beamline that allow for the beam spot to be focused down to a few mm^2. The beamline can be instrumented with a variety of beam monitoring detectors, collimators, and beam current measurement devices to precisely control the irradiation conditions. The facility also hosts a well equipped laboratory dedicated to the characterisation of samples after irradiation. An experimental validation of the irradiation setup, with proton fluxes ranging from 5×10^9 cm^-2s^-1 to 4×10^11 cm^-2s^-1, is reported

Intermittent C1-Inhibitor Deficiency Associated with Recessive Inheritance: Functional and Structural Insight

C1-inhibitor is a serine protease inhibitor (serpin) controlling complement and contact system activation. Gene mutations result in reduced C1-inhibitor functional plasma level causing hereditary angioedema, a life-threatening disorder. Despite a stable defect, the clinical expression of hereditary angioedema is unpredictable, and the molecular mechanism underlying this variability remains undisclosed. Here we report functional and structural studies on the Arg378Cys C1-inhibitor mutant found in a patient presenting reduced C1-inhibitor levels, episodically undergoing normalization. Expression studies resulted in a drop in mutant C1-innhibitor secretion compared to wild-type. Notwithstanding, the purified proteins had similar features. Thermal denaturation experiments showed a comparable denaturation profile, but the mutant thermal stability decays when tested in conditions reproducing intracellular crowding.Our findings suggest that once correctly folded, the Arg378Cys C1-inhibitor is secreted as an active, although quite unstable, monomer. However, it could bear a folding defect, occasionally promoting protein oligomerization and interfering with the secretion process, thus accounting for its plasma level variability. This defect is exacerbated by the nature of the mutation since the acquired cysteine leads to the formation of non-functional homodimers through inter-molecular disulphide bonding. All the proposed phenomena could be modulated by specific environmental conditions, rendering this mutant exceptionally vulnerable to mild stress