Investigating the Potential and Pitfalls of EV-Encapsulated MicroRNAs as Circulating Biomarkers of Breast Cancer

Extracellular vesicles (EVs) shuttle microRNA (miRNA) throughout the circulation and are believed to represent a fingerprint of the releasing cell. We isolated and characterized serum EVs of breast tumour-bearing animals, breast cancer (BC) patients, and healthy controls. EVs were characterized using transmission electron microscopy (TEM), protein quantification, western blotting, and nanoparticle tracking analysis (NTA). Absolute quantitative (AQ)-PCR was employed to analyse EV-miR-451a expression. Isolated EVs had the appropriate morphology and size. Patient sera contained significantly more EVs than did healthy controls. In tumour-bearing animals, a correlation between serum EV number and tumour burden was observed. There was no significant relationship between EV protein yield and EV quantity determined by NTA, highlighting the requirement for direct quantification. Using AQ-PCR to relate miRNA copy number to EV yield, a significant increase in miRNA-451a copies/EV was detected in BC patient sera, suggesting potential as a novel biomarker of breast cancer

A Trait‐Based Framework for Assessing the Vulnerability of Marine Species to Human Impacts

Marine species and ecosystems are widely affected by anthropogenic stressors, ranging from pollution and fishing to climate change. Comprehensive assessments of how species and ecosystems are impacted by anthropogenic stressors are critical for guiding conservation and management investments. Previous global risk or vulnerability assessments have focused on marine habitats, or on limited taxa or specific regions. However, information about the susceptibility of marine species across a range of taxa to different stressors everywhere is required to predict how marine biodiversity will respond to human pressures. We present a novel framework that uses life-history traits to assess species’ vulnerability to a stressor, which we compare across more than 44,000 species from 12 taxonomic groups (classes). Using expert elicitation and literature review, we assessed every combination of each of 42 traits and 22 anthropogenic stressors to calculate each species’ or representative species group’s sensitivity and adaptive capacity to stressors, and then used these assessments to derive their overall relative vulnerability. The stressors with the greatest potential impact were related to biomass removal (e.g., fisheries), pollution, and climate change. The taxa with the highest vulnerabilities across the range of stressors were mollusks, corals, and echinoderms, while elasmobranchs had the highest vulnerability to fishing-related stressors. Traits likely to confer vulnerability to climate change stressors were related to the presence of calcium carbonate structures, and whether a species exists across the interface of marine, terrestrial, and atmospheric realms. Traits likely to confer vulnerability to pollution stressors were related to planktonic state, organism size, and respiration. Such a replicable, broadly applicable method is useful for informing ocean conservation and management decisions at a range of scales, and the framework is amenable to further testing and improvement. Our framework for assessing the vulnerability of marine species is the first critical step toward generating cumulative human impact maps based on comprehensive assessments of species, rather than habitats

Genomic attributes of airway commensal bacteria and mucosa

Microbial communities at the airway mucosal barrier are conserved and highly ordered, in likelihood reflecting co-evolution with human host factors. Freed of selection to digest nutrients, the airway microbiome underpins cognate management of mucosal immunity and pathogen resistance. We show here the initial results of systematic culture and whole-genome sequencing of the thoracic airway bacteria, identifying 52 novel species amongst 126 organisms that constitute 75% of commensals typically present in heathy individuals. Clinically relevant genes encode antimicrobial synthesis, adhesion and biofilm formation, immune modulation, iron utilisation, nitrous oxide (NO) metabolism and sphingolipid signalling. Using whole-genome content we identify dysbiotic features that may influence asthma and chronic obstructive pulmonary disease. We match isolate gene content to transcripts and metabolites expressed late in airway epithelial differentiation, identifying pathways to sustain host interactions with microbiota. Our results provide a systematic basis for decrypting interactions between commensals, pathogens, and mucosa in lung diseases of global significance

Quantum state preparation and macroscopic entanglement in gravitational-wave detectors

Long-baseline laser-interferometer gravitational-wave detectors are operating at a factor of 10 (in amplitude) above the standard quantum limit (SQL) within a broad frequency band. Such a low classical noise budget has already allowed the creation of a controlled 2.7 kg macroscopic oscillator with an effective eigenfrequency of 150 Hz and an occupation number of 200. This result, along with the prospect for further improvements, heralds the new possibility of experimentally probing macroscopic quantum mechanics (MQM) - quantum mechanical behavior of objects in the realm of everyday experience - using gravitational-wave detectors. In this paper, we provide the mathematical foundation for the first step of a MQM experiment: the preparation of a macroscopic test mass into a nearly minimum-Heisenberg-limited Gaussian quantum state, which is possible if the interferometer's classical noise beats the SQL in a broad frequency band. Our formalism, based on Wiener filtering, allows a straightforward conversion from the classical noise budget of a laser interferometer, in terms of noise spectra, into the strategy for quantum state preparation, and the quality of the prepared state. Using this formalism, we consider how Gaussian entanglement can be built among two macroscopic test masses, and the performance of the planned Advanced LIGO interferometers in quantum-state preparation

Searching for a Stochastic Background of Gravitational Waves with LIGO

The Laser Interferometer Gravitational-wave Observatory (LIGO) has performed the fourth science run, S4, with significantly improved interferometer sensitivities with respect to previous runs. Using data acquired during this science run, we place a limit on the amplitude of a stochastic background of gravitational waves. For a frequency independent spectrum, the new limit is $\Omega_{\rm GW} < 6.5 \times 10^{-5}$. This is currently the most sensitive result in the frequency range 51-150 Hz, with a factor of 13 improvement over the previous LIGO result. We discuss complementarity of the new result with other constraints on a stochastic background of gravitational waves, and we investigate implications of the new result for different models of this background.Comment: 37 pages, 16 figure

Membrane-Anchored HIV-1 N-Heptad Repeat Peptides Are Highly Potent Cell Fusion Inhibitors via an Altered Mode of Action

Peptide inhibitors derived from HIV-gp41 envelope protein play a pivotal role in deciphering the molecular mechanism of HIV-cell fusion. According to accepted models, N-heptad repeat (NHR) peptides can bind two targets in an intermediate fusion conformation, thereby inhibiting progression of the fusion process. In both cases the orientation towards the endogenous intermediate conformation should be important. To test this, we anchored NHR to the cell membrane by conjugating fatty acids with increasing lengths to the N- or C-terminus of N36, as well as to two known N36 mutants; one that cannot bind C-heptad repeat (CHR) but can bind NHR (N36 MUTe,g), and the second cannot bind to either NHR or CHR (N36 MUTa,d). Importantly, the IC50 increased up to 100-fold in a lipopeptide-dependent manner. However, no preferred directionality was observed for the wild type derived lipopeptides, suggesting a planar orientation of the peptides as well as the endogenous NHR region on the cell membrane. Furthermore, based on: (i) specialized analysis of the inhibition curves, (ii) the finding that N36 conjugates reside more on the target cells that occupy the receptors, and (iii) the finding that N36 MUTe,g acts as a monomer both in its soluble form and when anchored to the cell membrane, we suggest that anchoring N36 to the cell changes the inhibitory mode from a trimer which can target both the endogenous NHR and CHR regions, to mainly monomeric lipopetides that target primarily the internal NHR. Besides shedding light on the mode of action of HIV-cell fusion, the similarity between functional regions in the envelopes of other viruses suggests a new approach for developing potent HIV-1 inhibitors

A Soluble Form of the High Affinity IgE Receptor, Fc-Epsilon-RI, Circulates in Human Serum

Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses. We here characterize a novel soluble version of the IgE-binding alpha-chain of Fc-epsilon-RI (sFcεRI), the high affinity receptor for IgE. sFcεRI immunoprecipitates as a protein of ∼40 kDa and contains an intact IgE-binding site. In human serum, sFcεRI is found as a soluble free IgE receptor as well as a complex with IgE. Using a newly established ELISA, we show that serum sFcεRI levels correlate with serum IgE in patients with elevated IgE. We also show that serum of individuals with normal IgE levels can be found to contain high levels of sFcεRI. After IgE-antigen-mediated crosslinking of surface FcεRI, we detect sFcεRI in the exosome-depleted, soluble fraction of cell culture supernatants. We further show that sFcεRI can block binding of IgE to FcεRI expressed at the cell surface. In summary, we here describe the alpha-chain of FcεRI as a circulating soluble IgE receptor isoform in human serum

Crop Updates 2002 - Farming Systems

This session covers forty one papers from different authors: INTRODUCTION 1. Future Farming Systems session for Crop Updates 2002 Peter Metcalf, FARMING SYSTEMS SUBPROGRAM MANAGER GRAINS PROGRAM Department of Agriculture 2. Perennial pastures in annual cropping systems: Lucerne and beyond, the ‘Big Picture’, Mike Ewing, Deputy CEO CRC for Plant-based Management of Dryland Salinity, Department of Agriculture 3. Perennial pastures in annual cropping systems: lucerne and beyond, Roy Latta and Keith Devenish, Department of Agriculture 4. Establishing Lucerne with a cover crop, Diana Fedorenko1, Clayton Butterly1, Chantelle Butterly1, Kim and Neil Diamond2, Stuart McAlpine2, Bill Bowden1, Jessica Johns3, 1Centre for Cropping Systems, Northam, 2Farmer, Buntine, 3Department of Agriculture 5. Overcropping: Chemical suppression of Lucerne, Terry Piper1, Diana Fedorenko1, Clayton Butterly1, Chantelle Butterly1, Stuart McAlpine2, Jessica Johns3, 1Centre for Cropping Systems, Northam, 2Farmer, Buntine, 3Department of Agriculture 6. Overcropping: Effect of Lucerne density on crop yield, Diana Fedorenko1, Bill Bowden1, Clayton Butterly1, Chantelle Butterly1, Stuart McAlpine2, Terry Piper1,1Centre for Cropping Systems, Department of Agriculture, Northam, 2Farmer, Buntine 7. Residual effect of weed management in the third year of Lucerne on the following wheat crop, Diana Fedorenko1, Clayton Butterly1, Chantelle Butterly1, Stuart McAlpine2,Terry Piper1, David Bowran1, Jessica Johns3,1Centre for Cropping Systems, Northam, 2Farmer, Buntine, 3Department of Agriculture 8. Production of Lucerne and serradella in four soil types, Diana Fedorenko1 Clayton Butterly1, Chantelle Butterly1, Robert Beard2 1Centre for Cropping Systems, Department of Agriculture, 2Farmer, Cunderdin 9. The effect of spray topping on newly established Lucerne, Keith Devenish, Agriculture Western Australia 10. Leakage from phase rotations involving Lucerne, Phil Ward, CSIRO Plant Industry 11. Fungal diseases present in Western Australian Lucerne crops, Dominie Wright and Nichole Burges, Department of Agriculture 12. Survey of Western Australian Lucerne stands reveals widespread virus infection, Roger Jones and Danae Harman, Crop Improvement Institute, Department of Agriculture, and Centre for Legumes in Mediterranean Agriculture, University of WA ANNUAL PASTURE SYSTEMS 13. The use of Twist Fungus as a biosecurity measure against Annual Ryegrass Toxicity (ARGT), Greg Shea, GrainGuard Coordinator and George Yan, Biological and Resource Technology 14.Limitations and opportunities for increasing water use by annual crops and pastures, David Tennant1, Phil Ward2and David Hall1 1Department of Agriculture, 2CSIRO, Plant Industries, Floreat Park 15. Developing pasture species mixtures for more productive and sustainable cropping systems – 2001 crop performance, Anyou Liu, Clinton Revell and Candy Hudson, Centre for Cropping Systems, Department of Agriculture 16. Developing pasture species mixtures for more productive and sustainable cropping systems – weed management in regenerating mixtures, Anyou Liu and Clinton Revell, Centre for Cropping Systems, Department of Agriculture 17. Aphid tolerance of annual pasture legumes, Andrew Blake, Natalie Lauritsen, Department of Agriculture 18. Selecting the right variety for phase pasture systems, Keith Devenish, Department of Agriculture 19. Responses of alternative annual pasture and forage legumes to challenge with infectious subterranean clover mottle virus, John Fosu-Nyarko, Roger Jones, Lisa Smith, Mike Jones and Geoff Dwyer, State Agricultural Biotechnology Centre and Centre for Bioinformatics and Biological Computing, Murdoch University, Department of Agriculture, and Centre for Legumes in Mediterranean Agriculture SOIL AND LAND MANAGEMENT 20. Nutrition in 2002: Decisions to be made as a result of last season, Bill Bowden,Western Australia Department of Agriculture 21. Profitability of deep banding lime, Michael O\u27Connell, Chris Gazey and David Gartner, Department of Agriculture 22. Lime efficiency percentage…the new measure of lime effectiveness for Western Australia, Amanda Miller, Department of Agriculture 23. Boron – should we be worried about it, Richard W. BellA, K. FrostA, Mike WongBand Ross BrennanC ASchool of Environmental Science, Murdoch University, BCSIRO Land and Water, CDepartment of Agriculture 24. Impact of claying and other amelioration on paddock profit, N.J. Blake1, G. McConnell2, D. Patabendige1and N. Venn11Department of Agriculture, 2PlanFarm P/L 25. Raised bed farming in the 2001 growing season, Derk Bakker, Greg Hamilton, Dave Houlbrooke and Cliff Spann, Department of Agriculture 26. Economics of tramline farming systems, Paul Blackwell and Bindi Webb, Department of Agriculture, Stuart McAlpine, Liebe Group. 27. Relay planting from Tramlines to increase water use and productivity os summer crops, Dr Paul Blackwell, Department of Agriculture, Neil and Kim Diamond, Buntine. Liebe Group 28.Evidence-based zone management of paddock variability to improve profits and environmental outcomes, M.T.F. WongA, D. PatabendigeB, G. LyleA and K. WittwerA ACSIRO Land and Water, BDepartment of Agriculture 29. How much soil water is lost over summer in sandy soils? Perry Dolling1, Senthold Asseng2, Ian Fillery2, Phil Ward2and Michael Robertson3 1University of Western Australia/Department of Agriculture Western Australia/CSIRO, 2CSIRO Plant Industry 3CSIRO Sustainable Ecosystems, Indooroopilly, Queensland FARMER DECISION SUPPORT AND ADOPTION 30. Economic comparisons of farming systems for the medium rainfall northern sandplain, No 1, Caroline Peek and David Rogers, Department of Agriculture 31. Sensitivity analysis of farming systems for the medium rainfall northern sandplain No 2, Caroline Peek and David Rogers, Department of Agriculture 32. Transition analysis of farming systems in the medium rainfall northern sandplain. No 3, Caroline Peek and David Rogers, Department of Agriculture 33. Implementing on-farm quality assurance, Peter Portmann, Manager Research and Development, The Grain Pool of Western Australia 34. On-farm research – principles of the ‘Test As You Grow’ kit, Jeff Russell, Department of Agriculture 35. Broadscale wheat variety comparisons featuring Wyalkatchem, Jeff Russell, Department of Agriculture 36. GrainGuardÔ - A biosecurity plan for the Canola Industry,Greg Shea Department of Agriculture 37. Are Western Australian broadacre farms efficient? Ben Henderson, University of Western Australia, Ross Kingwell, Department of Agriculture and University of Western Australia DISEASE MODELLING WORKSHOP 38. WORKSHOP: Pest and disease forecasts for you! An interactive forum, Tresslyn Walmsley, Jean Galloway, Debbie Thackray, Moin Salam and Art Diggle, Centre for Legumes in Mediterranean Agriculture and Department of Agriculture 39. Blackspot spread: Disease models are based in reality (Workshop paper 1), JeanGalloway,Department of Agriculture 40. Blackspot spread: Scaling-up field data to simulate ‘Baker’s farm’ (Workshop paper 2), Moin U. Salam, Jean Galloway, Art J. Diggle and William J. MacLeod, Department of Agriculture, Western Australia 41. A decision support system for control of aphids and CMV in lupin crops (Workshop paper 3), Debbie Thackray, Jenny Hawkes and Roger Jones, Centre for Legumes in Mediterranean Agriculture and Department of Agricultur

Direct Functionalization of Nitrogen Heterocycles via Rh-Catalyzed C−H Bond Activation

Nitrogen heterocycles are present in many compounds of enormous practical importance, ranging from pharmaceutical agents and biological probes to electroactive materials. Direct functionalization of nitrogen heterocycles through C−H bond activation constitutes a powerful means of regioselectively introducing a variety of substituents with diverse functional groups onto the heterocycle scaffold. Working together, our two groups have developed a family of Rh-catalyzed heterocycle alkylation and arylation reactions that are notable for their high level of functional-group compatibility. This Account describes our work in this area, emphasizing the relevant mechanistic insights that enabled synthetic advances and distinguished the resulting transformations from other methods. We initially discovered an intramolecular Rh-catalyzed C-2 alkylation of azoles by alkenyl groups. That reaction provided access to a number of di-, tri-, and tetracyclic azole derivatives. We then developed conditions that exploited microwave heating to expedite these reactions. While investigating the mechanism of this transformation, we discovered that a novel substrate-derived Rh−N-heterocyclic carbene (NHC) complex was involved as an intermediate. We then synthesized analogous Rh−NHC complexes directly by treating precursors to the intermediate [RhCl(PCy3)2] with N-methylbenzimidazole, 3-methyl-3,4-dihydroquinazoline, and 1-methyl-1,4-benzodiazepine-2-one. Extensive kinetic analysis and DFT calculations supported a mechanism for carbene formation in which the catalytically active RhCl(PCy3)2 fragment coordinates to the heterocycle before intramolecular activation of the C−H bond occurs. The resulting Rh−H intermediate ultimately tautomerizes to the observed carbene complex. With this mechanistic information and the discovery that acid cocatalysts accelerate the alkylation, we developed conditions that efficiently and intermolecularly alkylate a variety of heterocycles, including azoles, azolines, dihydroquinazolines, pyridines, and quinolines, with a wide range of functionalized olefins. We demonstrated the utility of this methodology in the synthesis of natural products, drug candidates, and other biologically active molecules. In addition, we developed conditions to directly arylate these heterocycles with aryl halides. Our initial conditions that used PCy3 as a ligand were successful only for aryl iodides. However, efforts designed to avoid catalyst decomposition led to the development of ligands based on 9-phosphabicyclo[4.2.1]nonane (phoban) that also facilitated the coupling of aryl bromides. We then replicated the unique coordination environment, stability, and catalytic activity of this complex using the much simpler tetrahydrophosphepine ligands and developed conditions that coupled aryl bromides bearing diverse functional groups without the use of a glovebox or purified reagents. With further mechanistic inquiry, we anticipate that researchers will better understand the details of the aforementioned Rh-catalyzed C−H bond functionalization reactions, resulting in the design of more efficient and robust catalysts, expanded substrate scope, and new transformations