Low-coverage massively parallel pyrosequencing of cDNAs enables proteomics in non-model species: Comparison of a species-specific database generated by pyrosequencing with databases from related species for proteome analysis of pea chloroplast envelopes

Bräutigam A, Shrestha RP, Whitten D, et al. Low-coverage massively parallel pyrosequencing of cDNAs enables proteomics in non-model species: Comparison of a species-specific database generated by pyrosequencing with databases from related species for proteome analysis of pea chloroplast envelopes. Journal of Biotechnology. 2008;136(1-2):44-53.Proteomics is a valuable tool for establishing and comparing the protein content of defined tissues, cell types, or subcellular structures. Its use in non-model species is currently limited because the identification of peptides Critically depends on sequence databases. In this study, we explored the potential of a preliminary cDNA database for the non-model species Pisum sativum created by a small number of massively parallel pyrosequencing (MPSS) runs for its use in proteomics and compared it to comprehensive cDNA databases from Medicago truncatula and Arabidopsis thaliana created by Sanger sequencing. Each database was used to identify Proteins from a pea leaf chloroplast envelope preparation. It is shown that the pea database identified more proteins with higher accuracy, although the sequence quality was low and the sequence contigs were short compared to databases from model species. Although the number of identified proteins in non-species-specific databases could potentially be increased by lowering the threshold for Successful protein identifications, this strategy markedly increases the number of wrongly identified proteins. The identification rate with non-species-specific databases correlated with spectral abundance but not with the predicted membrane helix content, and Strong conservation is necessary but not sufficient for protein identification with a non-species-specific database. It is concluded that massively Parallel sequencing of cDNAs substantially increases the power Of proteomics in non-model species. (C) 2008 Elsevier B.V. All rights reserved

Multidimensional collaboration; reflections on action research in a clinical context

This paper reflects on the challenges and benefits of multidimensional collaboration in an action research study to evaluate and improve preoperative education for patients awaiting colorectal surgery. Three cycles of planning, acting,observing and reflecting were designed to evaluate practice and implement change in this interactive setting, calling for specific and distinct collaborations. Data collection includes: observing educational interactions; administering patient evaluation questionnaires; interviewing healthcare staff, patients and carers; patient and carer focus groups; and examining written and audiovisual educational materials. The study revolves around and depends on multi-dimensional collaborations. Reflecting on these collaborations highlights the diversity of perspectives held by all those engaged in the study and enhances the action research lessons. Successfully maintaining the collaborations recognises the need for negotiation, inclusivity, comprehension, brokerage,and problem-solving. Managing the potential tensions is crucial to the successful implementation of changes introduced to practice and thus has important implications for patients’ well-being. This paper describes the experiences from an action research project involving new and specific collaborations, focusing on a particular healthcare setting. It exemplifies the challenges of the collaborative action research process and examines how both researchers and practitioners might reflect on the translation of theory into educational practices within a hospital colorectal department. Despite its context-specific features, the reflections on the types of challenges faced and lessons learned provide implications for action researchers in diverse healthcare settings across the world

Development and simulation of multi-diagnostic Bayesian analysis for 2D inference of divertor plasma characteristics

We present results of the design, implementation and testing of a Bayesian multi-diagnostic inference system which combines various divertor diagnostics to infer the 2D fields of electron temperature T e, density n e and deuterium neutral density n 0 in the divertor. The system was tested using synthetic diagnostic measurements derived from SOLPS-ITER fluid code predictions of the MAST-U Super-X divertor which include appropriate added noise. Two SOLPS-ITER simulations in different states of detachment, taken from a scan of the nitrogen seeding rate, were used as test-cases. Taken across both test-cases, the median absolute fractional errors in the inferred electron temperature and density estimates were 10.3% and 10.1% respectively. Differences between the inferred fields and the test-cases were well explained by solution uncertainty estimates derived from posterior sampling. This work represents a step toward a larger goal of obtaining a quantitative, 2D description of the divertor plasma state directly from experimental data, which could be used to gain better understanding of divertor physics phenomena

A neuron-specific cytoplasmic dynein isoform preferentially transports TrkB signaling endosomes

Cytoplasmic dynein is the multisubunit motor protein for retrograde movement of diverse cargoes to microtubule minus ends. Here, we investigate the function of dynein variants, defined by different intermediate chain (IC) isoforms, by expressing fluorescent ICs in neuronal cells. Green fluorescent protein (GFP)–IC incorporates into functional dynein complexes that copurify with membranous organelles. In living PC12 cell neurites, GFP–dynein puncta travel in both the anterograde and retrograde directions. In cultured hippocampal neurons, neurotrophin receptor tyrosine kinase B (TrkB) signaling endosomes are transported by cytoplasmic dynein containing the neuron-specific IC-1B isoform and not by dynein containing the ubiquitous IC-2C isoform. Similarly, organelles containing TrkB isolated from brain by immunoaffinity purification also contain dynein with IC-1 but not IC-2 isoforms. These data demonstrate that the IC isoforms define dynein populations that are selectively recruited to transport distinct cargoes

The Relative Contribution of Methanotrophs to Microbial Communities and Carbon Cycling in Soil Overlying a Coal-Bed Methane Seep

Seepage of coal-bed methane (CBM) through soils is a potential source of atmospheric CH4 and also a likely source of ancient (i.e. 14C-dead) carbon to soil microbial communities. Natural abundance 13C and 14C compositions of bacterial membrane phospholipid fatty acids (PLFAs) and soil gas CO2 and CH4 were used to assess the incorporation of CBM-derived carbon into methanotrophs and other members of the soil microbial community. Concentrations of type I and type II methanotroph PLFA biomarkers (16:1ω8c and 18:1ω8c, respectively) were elevated in CBM-impacted soils compared with a control site. Comparison of PLFA and 16s rDNA data suggested type I and II methanotroph populations were well estimated and overestimated by their PLFA biomarkers, respectively. The δ13C values of PLFAs common in type I and II methanotrophs were as negative as -67‰ and consistent with the assimilation of CBM. PLFAs more indicative of nonmethanotrophic bacteria had δ13C values that were intermediate indicating assimilation of both plantand CBM-derived carbon. Δ14C values of select PLFAs (-351 to -936‰) indicated similar patterns of CBM assimilation by methanotrophs and nonmethanotrophs and were used to estimate that 35–91% of carbon assimilated by nonmethanotrophs was derived from CBM depending on time of sampling and soil depth

A Comparison of Global Estimates of Marine Primary Production From Ocean Color

The third primary production algorithm round robin (PPARR3) compares output from 24 models that estimate depth-integrated primary production from satellite measurements of ocean color, as well as seven general circulation models (GCMs) coupled with ecosystem or biogeochemical models. Here we compare the global primary production fields corresponding to eight months of 1998 and 1999 as estimated from common input fields of photosynthetically-available radiation (PAR), sea-surface temperature (SST), mixed-layer depth, and chlorophyll concentration. We also quantify the sensitivity of the ocean-color-based models to perturbations in their input variables. The pair-wise correlation between ocean-color models was used to cluster them into groups or related output, which reflect the regions and environmental conditions under which they respond differently. The groups do not follow model complexity with regards to wavelength or depth dependence, though they are related to the manner in which temperature is used to parameterize photosynthesis. Global average PP varies by a factor of two between models. The models diverged the most for the Southern Ocean, SST under 10 degrees C, and chlorophyll concentration exceeding 1 mg Chl m-3. Based on the conditions under which the model results diverge most, we conclude that current ocean-color-based models are challenged by high-nutrient low-chlorophyll conditions, and extreme temperatures or chlorophyll concentrations. The GCM-based models predict comparable primary production to those based on ocean color: they estimate higher values in the Southern Ocean, at low SST, and in the equatorial band, while they estimate lower values in eutrophic regions (probably because the area of high chlorophyll concentrations is smaller in the GCMs). Further progress in primary production modeling requires improved understanding of the effect of temperature on photosynthesis and better parameterization of the maximum photosynthetic rate

An mRNA Blueprint for C-4 Photosynthesis Derived from Comparative Transcriptomics of Closely Related C-3 and C-4 Species

Bräutigam A, Kajala K, Wullenweber J, et al. An mRNA Blueprint for C-4 Photosynthesis Derived from Comparative Transcriptomics of Closely Related C-3 and C-4 Species. Plant Physiology. 2011;155(1):142-156.C-4 photosynthesis involves alterations to the biochemistry, cell biology, and development of leaves. Together, these modifications increase the efficiency of photosynthesis, and despite the apparent complexity of the pathway, it has evolved at least 45 times independently within the angiosperms. To provide insight into the extent to which gene expression is altered between C-3 and C-4 leaves, and to identify candidates associated with the C-4 pathway, we used massively parallel mRNA sequencing of closely related C-3 (Cleome spinosa) and C-4 (Cleome gynandra) species. Gene annotation was facilitated by the phylogenetic proximity of Cleome and Arabidopsis (Arabidopsis thaliana). Up to 603 transcripts differ in abundance between these C-3 and C-4 leaves. These include 17 transcription factors, putative transport proteins, as well as genes that in Arabidopsis are implicated in chloroplast movement and expansion, plasmodesmatal connectivity, and cell wall modification. These are all characteristics known to alter in a C-4 leaf but that previously had remained undefined at the molecular level. We also document large shifts in overall transcription profiles for selected functional classes. Our approach defines the extent to which transcript abundance in these C-3 and C-4 leaves differs, provides a blueprint for the NAD-malic enzyme C-4 pathway operating in a dicotyledon, and furthermore identifies potential regulators. We anticipate that comparative transcriptomics of closely related species will provide deep insight into the evolution of other complex traits

Existence and stability of viscoelastic shock profiles

We investigate existence and stability of viscoelastic shock profiles for a class of planar models including the incompressible shear case studied by Antman and Malek-Madani. We establish that the resulting equations fall into the class of symmetrizable hyperbolic--parabolic systems, hence spectral stability implies linearized and nonlinear stability with sharp rates of decay. The new contributions are treatment of the compressible case, formulation of a rigorous nonlinear stability theory, including verification of stability of small-amplitude Lax shocks, and the systematic incorporation in our investigations of numerical Evans function computations determining stability of large-amplitude and or nonclassical type shock profiles.Comment: 43 pages, 12 figure

Reconstituted B cell receptor signaling reveals carbohydrate-dependent mode of activation

Activation of immune cells (but not B cells) with lectins is widely known. We used the structurally defined interaction between influenza hemagglutinin (HA) and its cell surface receptor sialic acid (SA) to identify a B cell receptor (BCR) activation modality that proceeded through non-cognate interactions with antigen. Using a new approach to reconstitute antigen-receptor interactions in a human reporter B cell line, we found that sequence-defined BCRs from the human germline repertoire could be triggered by both complementarity to influenza HA and a separate mode of signaling that relied on multivalent ligation of BCR sialyl-oligosaccharide. The latter suggested a new mechanism for priming naïve B cell responses and manifested as the induction of SA-dependent pan-activation by peripheral blood B cells. BCR crosslinking in the absence of complementarity is a superantigen effect induced by some microbial products to subvert production of antigen-specific immune responses. B cell superantigen activity through affinity for BCR carbohydrate is discussed

The Far-Ultraviolet "Continuum" in Protoplanetary Disk Systems II: CO Fourth Positive Emission and Absorption

We exploit the high sensitivity and moderate spectral resolution of the $HST$-Cosmic Origins Spectrograph to detect far-ultraviolet spectral features of carbon monoxide (CO) present in the inner regions of protoplanetary disks for the first time. We present spectra of the classical T Tauri stars HN Tau, RECX-11, and V4046 Sgr, representative of a range of CO radiative processes. HN Tau shows CO bands in absorption against the accretion continuum. We measure a CO column density and rotational excitation temperature of N(CO) = 2 +/- 1 $\times$ 10$^{17}$ cm$^{-2}$ and T_rot(CO) 500 +/- 200 K for the absorbing gas. We also detect CO A-X band emission in RECX-11 and V4046 Sgr, excited by ultraviolet line photons, predominantly HI LyA. All three objects show emission from CO bands at $\lambda$ $>$ 1560 \AA, which may be excited by a combination of UV photons and collisions with non-thermal electrons. In previous observations these emission processes were not accounted for due to blending with emission from the accretion shock, collisionally excited H$_{2}$, and photo-excited H2; all of which appeared as a "continuum" whose components could not be separated. The CO emission spectrum is strongly dependent upon the shape of the incident stellar LyA emission profile. We find CO parameters in the range: N(CO) 10$^{18-19}$ cm$^{-2}$, T_{rot}(CO) > 300 K for the LyA-pumped emission. We combine these results with recent work on photo- and collisionally-excited H$_{2}$ emission, concluding that the observations of ultraviolet-emitting CO and H2 are consistent with a common spatial origin. We suggest that the CO/H2 ratio in the inner disk is ~1, a transition between the much lower interstellar value and the higher value observed in solar system comets today, a result that will require future observational and theoretical study to confirm.Comment: 12 pages, 7 figures, 3 tables. ApJ - accepte