Lamellar body ultrastructure revisited: high-pressure freezing and cryo-electron microscopy of vitreous sections

Lamellar bodies are the storage sites for lung surfactant within type II alveolar epithelial cells. The structure-function models of lamellar bodies are based on microscopic analyses of chemically fixed tissue. Despite available alternative fixation methods that are less prone to artifacts, such as cryofixation by high-pressure freezing, the nature of the lung, being mostly air filled, makes it difficult to take advantage of these improved methods. In this paper, we propose a new approach and show for the first time the ultrastructure of intracellular lamellar bodies based on cryo-electron microscopy of vitreous sections in the range of nanometer resolution. Thus, unspoiled by chemical fixation, dehydration and contrasting agents, a close to native structure is revealed. Our approach uses perfluorocarbon to substitute the air in the alveoli. Lung tissue was subsequently high-pressure frozen, cryosectioned and observed in a cryo-electron microscope. The lamellar bodies clearly show a tight lamellar morphology. The periodicity of these lamellae was 7.3 nm. Lamellar bifurcations were observed in our cryosections. The technical approach described in this paper allows the examination of the native cellular ultrastructure of the surfactant system under near in vivo conditions, and therefore opens up prospectives for scrutinizing various theories of lamellar body biogenesis, exocytosis and recycling.

Lipid remodelling of glycosylphosphatidylinositol (GPI) glycoconjugates in procyclic-form trypanosomes: biosynthesis and processing of GPIs revisited

The African trypanosome, Trypanosoma brucei, has been used as a model to study the biosynthesis of GPI (glycosylphosphatidylinositol) anchors. In mammalian (bloodstream)-form parasites, diacyl-type GPI precursors are remodelled in their lipid moieties before attachment to variant surface glycoproteins. In contrast, the GPI precursors of insect (procyclic)-form parasites, consisting of lyso-(acyl)PI (inositol-acylated acyl-lyso-phosphatidylinositol) species, remain unaltered before protein attachment. By using a combination of metabolic labelling, cell-free assays and complementary MS analyses, we show in the present study that GPI-anchored glycoconjugates in T. congolense procyclic forms initially receive tri-acylated GPI precursors, which are subsequently de-acylated either at the glycerol backbone or on the inositol ring. Chemical and enzymatic treatments of [3H]myristate-labelled lipids in combination with ESI-MS/MS (electrospray ionization-tandem MS) and MALDI-QIT-TOF-MS3 (matrix-assisted laser-desorption ionization-quadrupole ion trap-time-of-flight MS) analyses indicate that the structure of the lipid moieties of steady-state GPI lipids from T. congolense procyclic forms consist of a mixture of lyso-(acyl)PI, diacyl-PI and diacyl-(acyl)PI species. Interestingly, some of these species are myristoylated at the sn-2 position. To our knowledge, this is the first demonstration of lipid remodelling at the level of protein- or polysaccharide-linked GPI anchors in procyclic-form trypanosomes.

Design, expression, and processing of epitomized hepatitis C virus-encoded CTL epitopes

Hepatitis C virus (HCV) vaccine efficacy may crucially depend on immunogen length and coverage of viral sequence diversity. However, covering a considerable proportion of the circulating viral sequence variants would likely require long immunogens, which for the conserved portions of the viral genome, would contain unnecessarily redundant sequence information. In this study, we present the design and in vitro performance analysis of a novel "epitome" approach that compresses frequent immune targets of the cellular immune response against HCV into a shorter immunogen sequence. Compression of immunological information is achieved by partial overlapping shared sequence motifs between individual epitopes. At the same time, sequence diversity coverage is provided by taking advantage of emerging cross-reactivity patterns among epitope variants so that epitope variants associated with the broadest variant cross-recognition are preferentially included. The processing and presentation analysis of specific epitopes included in such a compressed, in vitro-expressed HCV epitome indicated effective processing of a majority of tested epitopes, although re-presentation of some epitopes may require refined sequence design. Together, the present study establishes the epitome approach as a potential powerful tool for vaccine immunogen design, especially suitable for the induction of cellular immune responses against highly variable pathogens.

Integrated data acquisition and processing to determine metabolite contents, relaxation times, and macromolecule baseline in single examinations of individual subjects

Absolute quantitation of clinical (1)H-MR spectra is virtually always incomplete for single subjects because the separate determination of spectrum, baseline, and transverse and longitudinal relaxation times in single subjects is prohibitively long. Integrated Processing and Acquisition of Data (IPAD) based on a combined 2-dimensional experimental and fitting strategy is suggested to substantially improve the information content from a given measurement time. A series of localized saturation-recovery spectra was recorded and combined with 2-dimensional prior-knowledge fitting to simultaneously determine metabolite T(1) (from analysis of the saturation-recovery time course), metabolite T(2) (from lineshape analysis based on metabolite and water peak shapes), macromolecular baseline (based on T(1) differences and analysis of the saturation-recovery time course), and metabolite concentrations (using prior knowledge fitting and conventional procedures of absolute standardization). The procedure was tested on metabolite solutions and applied in 25 subjects (15-78 years old). Metabolite content was comparable to previously found values. Interindividual variation was larger than intraindividual variation in repeated spectra for metabolite content as well as for some relaxation times. Relaxation times were different for various metabolite groups. Parts of the interindividual variation could be explained by significant age dependence of relaxation times.

Perceptual and processing differences between physical and dichorhinic odor mixtures

Perceptual integration of sensory input from our two nostrils has received little attention in comparison to lateralized inputs for vision and hearing. Here, we investigated whether a binary odor mixture of eugenol and l-carvone (smells of cloves and caraway) would be perceived differently if presented as a mixture in one nostril (physical mixture), vs. the same two odorants in separate nostrils (dichorhinic mixture). In parallel, we investigated whether the different types of presentation resulted in differences in olfactory event-related potentials (OERP). Psychophysical ratings showed that the dichorhinic mixtures were perceived as more intense than the physical mixtures. A tendency for shift in perceived quality was also observed. In line with these perceptual changes, the OERP showed a shift in latencies and amplitudes for early (more "sensory") peaks P1 and N1 whereas no significant differences were observed for the later (more "cognitive") peak P2. The results altogether suggest that the peripheral level is a site of interaction between odorants. Both psychophysical ratings and, for the first time, electrophysiological measurements converge on this conclusion.

Digital Data Collection and Processing in Field and Lab: The Approach of Kinneret Regional Project

The paper showcases the field- and lab-documentation system developed for Kinneret Regional Project, an international archaeological expedition to the Northwestern shore of the Sea of Galilee (Israel) under the auspices of the University of Bern, the University of Helsinki, Leiden University and Wofford College. The core of the data management system is a fully relational, server-based database framework, which also includes time-based and static GIS services, stratigraphic analysis tools and fully indexed document/digital image archives. Data collection in the field is based on mobile, hand-held devices equipped with a custom-tailored stand-alone application. Comprehensive three-dimensional documentation of all finds and findings is achieved by means of total stations and/or high-precision GPS devices. All archaeological information retrieved in the field – including tachymetric data – is synched with the core system on the fly and thus immediately available for further processing in the field lab (within the local network) or for post-excavation analysis at remote institutions (via the WWW). Besides a short demonstration of the main functionalities, the paper also presents some of the key technologies used and illustrates usability aspects of the system’s individual components.

Influence of carbon enrichment on electrical conductivity and processing of polycarbosilane derived ceramic for MEMS applications

An analysis about the effect of carbon enrichment of allylhydridopolycarbosilane SMP10® with divinylbenzene (DVB) as a promising material for electrical conductive micro-electrical mechanical systems (MEMS) is presented. The liquid precursors can be micropipetted and cured in polytetrafluoroethylene (PTFE) molds to produce 14 mm diameter disc shaped samples. The effect of pyrolysis temperature and carbon content on the electrical conductivity is discussed. The addition of 28.7 wt.% of DVB was found to be the optimum amount. Carbon was preserved in the microstructure during pyrolysis and the ceramic yield increased from 77.5 to 80.5 wt.%. The electrical conductivity increased from 10−6 to 1 S/cm depending on the annealing temperature. Furthermore, the ceramic samples obtained with this composition were found to be in many cases crack free or with minimal cracks in contrast with the behavior of pure SMP10. Using the same process we demonstrate that also microsized ceramic samples can be produced.

The impact of absolute importance and processing overlaps on prospective memory performance

Prospective memory is the ability to remember an intention at an appropriate moment in the future. Prospective memory tasks can be more or less important. Previously, importance was manipulated by emphasizing the importance of the prospective memory task relative to the ongoing task it was embedded in. This resulted in better prospective memory performance but also ongoing task costs. In the present study, we simply instructed one group of participants that the prospective memory task was important (i.e., absolute importance instruction) and compared them to a group with relative importance instructions and a control group. The results showed that absolute importance lead to an increase in prospective memory performance without enhancing ongoing task costs, whereas relative importance resulted in both increased prospective memory performance and ongoing task costs. Thus, prospective memory can be enhanced without ongoing task costs, which is particularly crucial for safety-work contexts.

Expression and processing of Plasmodium berghei SERA3 during liver stages.

Cysteine proteases mediate liberation of Plasmodium berghei merozoites from infected hepatocytes. In an attempt to identify the responsible parasite proteases, we screened the genome of P. berghei for cysteine protease-encoding genes. RT-PCR analyses revealed that transcription of four out of five P. berghei serine repeat antigen (PbSERA) genes was strongly upregulated in late liver stages briefly before the parasitophorous vacuole membrane ruptured to release merozoites into the host cell cytoplasm, suggesting a role of PbSERA proteases in these processes. In order to characterize PbSERA3 processing, we raised an antiserum against a non-conserved region of the protein and generated a transgenic P. berghei strain expressing a TAP-tagged PbSERA3 under the control of the endogenous promoter. Immunofluorescence assays revealed that PbSERA3 leaks into the host cell cytoplasm during merozoite development, where it might contribute to host cell death or activate host cell proteases that execute cell death. Importantly, processed PbSERA3 has been detected by Western blot analysis in cell extracts of schizont-infected cells and merozoite-infected detached hepatic cells.

Ceramic formulation and processing design for plutonium disposition

The focus of this research programme is to develop a single phase ceramic wasteform for waste PuO2 that is unsuitable for fuel manufacture. A suite of synthetic mineral systems have been considered including titanate, zirconate, phosphate and silicate based matrices. Although a wealth of information on plutonium disposition in some of the systems exists in the literature, the data is not always directly comparable which hinders comparison between different ceramic hosts. The crux of this research has been to compile a database of information on the proposed hosts to allow impartial comparison of the relative merits of each system. © 2009 Materials Research Society.