Multi-scale three-dimensional characterization of iron particles in dusty olivine: Implications for paleomagnetism of chondritic meteorites

Dusty olivine (olivine containing multiple sub-micrometer inclusions of metallic iron) in chondritic meteorites is considered an ideal carrier of paleomagnetic remanence, capable of maintaining a faithful record of pre-accretionary magnetization acquired during chondrule formation. Here we show how the magnetic architecture of a single dusty olivine grain from the Semarkona LL3.0 ordinary chondrite meteorite can be fully characterised in three dimensions, using a combination of Focussed-Ion-Beam nanotomography (FIB-nT), electron tomography and finite-element micromagnetic modelling. We present a three-dimensional (3D) volume reconstruction of a dusty olivine grain, obtained by selective milling through a region of interest in a series of sequential 20 nm slices, which are then imaged using scanning electron microscopy. The data provide a quantitative description of the iron particle ensemble, including the distribution of particle sizes, shapes, interparticle spacings and orientations. Iron particles are predominantly oblate ellipsoids with average radii 242 ± 94 nm by 199 ± 80 nm by 123 ± 58 nm. Using analytical TEM we observe that the particles nucleate on sub-grain boundaries and are loosely arranged in a series of sheets parallel to (001) of the olivine host. This is in agreement with the orientation data collected using the FIB-nT, and highlights how the underlying texture of the dusty olivine is crystallographically constrained by the olivine host. The shortest dimension of the particles is oriented normal to the sheets and their longest dimension is preferentially aligned within the sheets. Individual particle geometries are converted to a finite-element mesh and used to perform micromagnetic simulations. The majority of particles adopt a single vortex state, with ‘bulk’ spins that rotate around a central vortex core. We observed no particles, which are in a true single domain state. The results of the micromagnetic simulations challenge some pre-conceived ideas about the remanence carrying properties of vortex states. There is often not a simple predictive relationship between the major, intermediate and minor axes of the particles and the remanence vector imparted in different fields. Although the orientation of the vortex core is determined largely by the ellipsoidal geometry (i.e., parallel to the major axis for prolate ellipsoids and parallel to the minor axis for oblate ellipsoids), the core and remanence vectors can sometimes lie at very large (tens of degree) angles to the principal axes. The subtle details of the morphology can control the overall remanence state, leading in some cases to a dominant contribution from the bulk spins to the net remanence, with profound implications for predicting the anisotropy of the sample. The particles have very high switching fields (several hundred mT), demonstrating their high stability and suitability for paleointensity studies.The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC grant agreements 291522-3DIMAGE (P.A.M.) and 320750 - Nanopaleomagnetism (J.F.E., R.J.H., and P.A.M.). BPW and RRF were supported by NASA Emerging Worlds program grant #NNX15AH72G, the NASA Solar System Exploration and Research Virtual Institute grant #NNA14AB01A, and a generous gift from Thomas F. Peterson, Jr. The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement No. 320832-Imagine. (W.W . and P.O.C.) W.W. was also supported for this research under NERC grant NE/J020966/1 - Predicting the reliability with which the geomagnetic field can be recorded in igneous rocks.This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by the Mineralogical Society of America

Protein interactions in Xenopus germ plasm RNP particles

Hermes is an RNA-binding protein that we have previously reported to be found in the ribonucleoprotein (RNP) particles of Xenopus germ plasm, where it is associated with various RNAs, including that encoding the germ line determinant Nanos1. To further define the composition of these RNPs, we performed a screen for Hermes-binding partners using the yeast two-hybrid system. We have identified and validated four proteins that interact with Hermes in germ plasm: two isoforms of Xvelo1 (a homologue of zebrafish Bucky ball) and Rbm24b and Rbm42b, both RNA-binding proteins containing the RRM motif. GFP-Xvelo fusion proteins and their endogenous counterparts, identified with antisera, were found to localize with Hermes in the germ plasm particles of large oocytes and eggs. Only the larger Xvelo isoform was naturally found in the Balbiani body of previtellogenic oocytes. Bimolecular fluorescence complementation (BiFC) experiments confirmed that Hermes and the Xvelo variants interact in germ plasm, as do Rbm24b and 42b. Depletion of the shorter Xvelo variant with antisense oligonucleotides caused a decrease in the size of germ plasm aggregates and loosening of associated mitochondria from these structures. This suggests that the short Xvelo variant, or less likely its RNA, has a role in organizing and maintaining the integrity of germ plasm in Xenopus oocytes. While GFP fusion proteins for Rbm24b and 42b did not localize into germ plasm as specifically as Hermes or Xvelo, BiFC analysis indicated that both interact with Hermes in germ plasm RNPs. They are very stable in the face of RNA depletion, but additive effects of combinations of antisense oligos suggest they may have a role in germ plasm structure and may influence the ability of Hermes protein to effectively enter RNP particles

A new ultrafast and high-throughput mass spectrometric approach for the therapeutic drug monitoring of the multi-targeted anti-folate pemetrexed in plasma from lung cancer patients

An analytical assay has been developed and validated for ultrafast and high-throughput mass spectrometric determination of pemetrexed concentrations in plasma using matrix assisted laser desorption/ionization–triple quadrupole–tandem mass spectrometry. Patient plasma samples spiked with the internal standard methotrexate were measured by multiple reaction monitoring. The detection limit was 0.4 fmol/μL, lower limit of quantification was 0.9 fmol/μL, and upper limit of quantification was 60 fmol/μL, respectively. Overall observed pemetrexed concentrations in patient samples ranged between 8.7 (1.4) and 142.7 (20.3) pmol/μL (SD). The newly developed mass spectrometric assay is applicable for (routine) therapeutic drug monitoring of pemetrexed concentrations in plasma from non-small cell lung cancer patients

Testing peatland testate amoeba transfer functions: Appropriate methods for clustered training-sets

Transfer functions are widely used in palaeoecology to infer past environmental conditions from fossil remains of many groups of organisms. In contrast to traditional training-set design with one observation per site, some training-sets, including those for peatland testate amoeba-hydrology transfer functions, have a clustered structure with many observations from each site. Here we show that this clustered design causes standard performance statistics to be overly optimistic. Model performance when applied to independent data sets is considerably weaker than suggested by statistical cross-validation. We discuss the reasons for these problems and describe leave-one-site-out cross-validation and the cluster bootstrap as appropriate methods for clustered training-sets. Using these methods we show that the performance of most testate amoeba-hydrology transfer functions is worse than previously assumed and reconstructions are more uncertain

Localisation of RNAs into the germ plasm of vitellogenic xenopus oocytes

We have studied the localisation of mRNAs in full-grown Xenopus laevis oocytes by injecting fluorescent RNAs, followed by confocal microscopy of the oocyte cortex. Concentrating on RNA encoding the Xenopus Nanos homologue, nanos1 (formerly Xcat2), we find that it consistently localised into aggregated germ plasm ribonucleoprotein (RNP) particles, independently of cytoskeletal integrity. This implies that a diffusion/entrapment-mediated mechanism is active, as previously reported for previtellogenic oocytes. Sometimes this was accompanied by localisation into scattered particles of the “late”, Vg1/VegT pathway; occasionally only late pathway localisation was seen. The Xpat RNA behaved in an identical fashion and for neither RNA was the localisation changed by any culture conditions tested. The identity of the labelled RNP aggregates as definitive germ plasm was confirmed by their inclusion of abundant mitochondria and co-localisation with the germ plasm protein Hermes. Further, the nanos1/Hermes RNP particles are interspersed with those containing the germ plasm protein Xpat. These aggregates may be followed into the germ plasm of unfertilized eggs, but with a notable reduction in its quantity, both in terms of injected molecules and endogenous structures. Our results conflict with previous reports that there is no RNA localisation in large oocytes, and that during mid-oogenesis even germ plasm RNAs localise exclusively by the late pathway. We find that in mid oogenesis nanos1 RNA also localises to germ plasm but also by the late pathway. Late pathway RNAs, Vg1 and VegT, also may localise into germ plasm. Our results support the view that mechanistically the two modes of localisation are extremely similar, and that in an injection experiment RNAs might utilise either pathway, the distinction in fates being very subtle and subject to variation. We discuss these results in relation to their biological significance and the results of others

A Highly Conserved Poc1 Protein Characterized in Embryos of the Hydrozoan Clytia hemisphaerica: Localization and Functional Studies

Poc1 (Protein of Centriole 1) proteins are highly conserved WD40 domain-containing centriole components, well characterized in the alga Chlamydomonas, the ciliated protazoan Tetrahymena, the insect Drosophila and in vertebrate cells including Xenopus and zebrafish embryos. Functions and localizations related to the centriole and ciliary axoneme have been demonstrated for Poc1 in a range of species. The vertebrate Poc1 protein has also been reported to show an additional association with mitochondria, including enrichment in the specialized “germ plasm” region of Xenopus oocytes. We have identified and characterized a highly conserved Poc1 protein in the cnidarian Clytia hemisphaerica. Clytia Poc1 mRNA was found to be strongly expressed in eggs and early embryos, showing a punctate perinuclear localization in young oocytes. Fluorescence-tagged Poc1 proteins expressed in developing embryos showed strong localization to centrioles, including basal bodies. Anti-human Poc1 antibodies decorated mitochondria in Clytia, as reported in human cells, but failed to recognise endogenous or fluorescent-tagged Clytia Poc1. Injection of specific morpholino oligonucleotides into Clytia eggs prior to fertilization to repress Poc1 mRNA translation interfered with cell division from the blastula stage, likely corresponding to when neosynthesis normally takes over from maternally supplied protein. Cell cycle lengthening and arrest were observed, phenotypes consistent with an impaired centriolar biogenesis or function. The specificity of the defects could be demonstrated by injection of synthetic Poc1 mRNA, which restored normal development. We conclude that in Clytia embryos, Poc1 has an essentially centriolar localization and function

Role of Germination in Murine Airway CD8+ T-Cell Responses to Aspergillus Conidia

Pulmonary exposure to Aspergillus fumigatus has been associated with morbidity and mortality, particularly in immunocompromised individuals. A. fumigatus conidia produce β-glucan, proteases, and other immunostimulatory factors upon germination. Murine models have shown that the ability of A. fumigatus to germinate at physiological temperature may be an important factor that facilitates invasive disease. We observed a significant increase in IFN-γ-producing CD8+ T cells in bronchoalveolar lavage fluid (BALF) of immunocompetent mice that repeatedly aspirated A. fumigatus conidia in contrast to mice challenged with A. versicolor, a species that is not typically associated with invasive, disseminated disease. Analysis of tissue sections indicated the presence of germinating spores in the lungs of mice challenged with A. fumigatus, but not A. versicolor. Airway IFN-γ+CD8+ T-cells were decreased and lung germination was eliminated in mice that aspirated A. fumigatus conidia that were formaldehyde-fixed or heat-inactivated. Furthermore, A. fumigatus particles exhibited greater persistence in the lungs of recipient mice when compared to non-viable A. fumigatus or A. versicolor, and this correlated with increased maintenance of airway memory-phenotype CD8+ T cells. Therefore, murine airway CD8+ T cell-responses to aspiration of Aspergillus conidia may be mediated in part by the ability of conidia to germinate in the host lung tissue. These results provide further evidence of induction of immune responses to fungi based on their ability to invade host tissue

The Impact of Simulated Sulfate Deposition on Peatland Testate Amoebae

Peatlands subjected to sulfate deposition have been shown to produce less methane, believed to be due to competitive exclusion of methanogenic archaea by sulfate-reducing bacteria. Here, we address whether sulfate deposition produces impacts on a higher microbial group, the testate amoebae. Sodium sulfate was applied to experimental plots on a Scottish peatland and samples extracted after a period of more than 10 years. Impacts on testate amoebae were tested using redundancy analysis and Mann-Whitney tests. Results showed statistically significant impacts on amoebae communities particularly noted by decreased abundance of Trinema lineare, Corythion dubium, and Euglypha rotunda. As the species most reduced in abundance are all small bacterivores we suggest that our results support the hypothesis of a shift in dominant prokaryotes, although other explanations are possible. Our results demonstrate the sensitivity of peatland microbial communities to sulfate deposition and suggest sulfate may be a potentially important secondary control on testate amoebae communities

Resident Memory T Cells (TRM) Are Abundant in Human Lung: Diversity, Function, and Antigen Specificity

Recent studies have shown that tissue resident memory T cells (TRM) are critical to antiviral host defense in peripheral tissues. This new appreciation of TRM that reside in epithelial tissues and mediate host defense has been studied most extensively in skin: adult human skin contains large numbers of functional TRM that express skin specific markers. Indeed, more than twice as many T cells reside in skin as in peripheral blood. This T cell population has a diverse T cell receptor repertoire, and can produce a broad array of cytokines. More recently, we have begun to examine other epithelial tissues for the presence of resident T cells. In the present study, we asked whether analogous populations of resident T cells could be found in human lung. We were able to demonstrate abundant resident T cells in human lung-more than 10 billion T cells were present. Lung T cells were largely of the effector memory T cell (TEM) phenotype, though small numbers of central memory T cells (TCM) and T regulatory cells (Treg) could be identified. Lung T cells had a diverse T cell receptor repertoire and subsets produced IL-17, IL-4, IFNγ, as well as TNFα. A significant number of lung TRM CD4+Th cells produced more than one cytokine, identifying them as “multifunctional” Th1 type cells. Finally, lung TRM, but not TRM resident to skin or T cells from blood, proliferated in response to influenza virus. This work suggests that normal human lung contains large numbers of TRM cells, and these cells are poised to respond to recall antigens previously encountered through lung mucosa. This population of T cells may contribute to the pathogenesis of asthma and other T cell mediated lung diseases

Rapid Reactivation of Extralymphoid CD4 T Cells during Secondary Infection

After infection, extralymphoid tissues are enriched with effector and memory T cells of a highly activated phenotype. The capacity for rapid effector cytokine response from extralymphoid tissue-memory T cells suggests these cells may perform a ‘sentinel’ function in the tissue. While it has been demonstrated that extralymphoid CD4+ T cells can directly respond to secondary infection, little is known about how rapidly this response is initiated, and how early activation of T cells in the tissue may affect the innate response to infection. Here we use a mouse model of secondary heterosubtypic influenza infection to show that CD4+ T cells in the lung airways are reactivated within 24 hours of secondary challenge. Airway CD4+ T cells initiate an inflammatory cytokine and chemokine program that both alters the composition of the early innate response and contributes to the reduction of viral titers in the lung. These results show that, unlike a primary infection, extralymphoid tissue-memory CD4+ T cells respond alongside the innate response during secondary infection, thereby shaping the overall immune profile in the airways. These data provide new insights into the role of extralymphoid CD4+ T cells during secondary immune responses